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DNA structures associated with the Fragile X triplet repeat sequences

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DNA structures associated with the Fragile X triplet repeat sequences

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Content DNA STRUCTURES ASSOCIATED WITH THE FRAGILE X TRIPLET REPEAT SEQUENCES by Pomchai Rojsitthisak A Dissertation Presented to the FACULTY OF THE GRADUATE SCHOOL UNIVERSITY OF SOUTHERN CALIFORNIA In Partial Fulfillment of the Requirements of the Degree DOCTOR OF PHILOSOPHY (PHARMACEUTICAL SCIENCES) August 2002 Copyright 2002 Pomchai Rojsitthisak Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. UMI Number: 3094372 UMI UMI Microform 3094372 Copyright 2003 by ProQuest Information and Learning Company. All rights reserved. This microform edition is protected against unauthorized copying under Title 17, United States Code. ProQuest Information and Learning Company 300 North Zeeb Road P.O. Box 1346 Ann Arbor, Ml 48106-1346 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. UNIVERSITY OF SOUTHERN CALIFORNIA The Graduate School University Park LOS ANGELES, CALIFORNIA 900894695 This dissertation, w ritten b y ROTSlTTVUSAK U nder th e direction o f h jl... D issertation Com m ittee, and approved b y a ll its m em bers, has been p resen ted to an d accepted b y The G raduate School, in p a rtia l fulfillm ent o f requirem ents fo r th e degree o f DOCTOR OF PHILOSOPHY Dean o f Graduate Studies D ate —A ugust 6„ 2Q 0 2— D ISSERJATIpN COMMITTEE Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. DEDICATION In loving memory of Boonlin Rojsitthisak His son Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. iii ACKNOWLEDGEMENTS I would like to express my deep gratitude to my Ph.D. advisor, Dr. Ian S. Haworth, for his valuable advice, continual guidance, kindness, and understanding throughout my graduate study. His scientific open-mindedness encouraged me to successfully complete this unique research. I am sincerely grateful to Dr. Michael Bolger, Dr. Sarah Hamm-Alvarez, Dr. Wei-Chiang Shen and Dr. Debbie Johnson for serving on my Ph.D. committee. Their valuable suggestions and discussions made my research more accurate. I would also like to thank Dr. Robert T. Koda for his generosity in permitting me to use the HPLC machine in his Pharmacokinetics Laboratory. In addition, I thank Dr. Rebecca M. Romero for sharing her exceptional technical skills, and for providing so much help. I would also like to acknowledge Chulalongkom University (Bangkok, Thailand) for their generosity in providing me with a scholarship, without which I could not have completed my doctoral studies. Furthermore, I am grateful to all of my colleagues at Dr. Haworth’s and Dr. Koda’s Laboratories for their encouragement. Finally, I am grateful to my beloved parents, brothers and sisters for their support throughout my graduate years. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. iv TABLE OF CONTENTS Dedication........................................................................................................................ii Acknowledgements....................................................................................................... iii List of Tables..................................................................................................................ix List of Figures................................................................................................................. x Abbreviations...............................................................................................................xiv Abstract............................................................................................................. xv Chapter I: Introduction................................................................................................ 1 1.1 Diseases Associated with DNA Triplet Repeat Expansion................................1 1.2 Triplet Repeat Expansion Diseases Type I ..........................................................4 1.3 Triplet Repeat Expansion Diseases Type II......................................................... 4 1.4 Alternative DNA Structures Formed in Triplet Repeats..................................... 5 1.4.1 Unusual Characteristics of DNA Duplexes Containing Triplet Repeats.. 6 1.4.2 Intramolecular Hairpin Structures in Single-Stranded d[CTG]n, d[CAG]n, d[CGG]n, and d[CCG]n................................................................7 1.4.3 Triplex DNA Structures in d[GAA]n»d[TTC]n...........................................9 1.4.4 Quadruplex DNA Structures in Single-Stranded d[CGG]n..................... 11 1.4.5 Slipped-Strand DNA Structures.................................................................12 1.5 Molecular Mechanism of Triplet Repeat Expansion/Deletion..........................13 1.5.1 Triplet Repeat Expansion/Deletion Caused by DNA Replication.......... 14 1.5.2 Triplet Repeat Expansion/Deletion Caused by DNA Repair.................. 20 1.5.3 Triplet Repeat Expansion/Deletion Caused by DNA Recombination... 22 1.6 Fragile X Syndrome............................................................................................. 24 1.7 Conformational Properties of Structures Formed by d[CCG]n........................ 27 1.8 Mechlorethamine DNA Crosslinking Reaction................................................. 30 1.9 Dissertation Overview..........................................................................................32 1.10 Dissertation Significance................................................................................... 34 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. V Chapter II: DNA Interstrand Crosslink Formation by Mechlorethamine at a Cytosine-Cytosine Mismatched Pair: Kinetics and Sequence Dependence...............................................................................35 2.1 Introduction........................................................................................................... 35 2.2 Materials and Methods......................................................................................... 37 2.3 Results................................................................................................................... 41 2.3.1 The mechlorethamine C-C crosslink forms more rapidly than the 1,3 G-G crosslink, and reaches a higher final yield................... 41 2.3.2 The mechlorethamine C-C crosslink is more stable than the 1,3 G-G crosslink..........................................................................44 2.3.3 The amount of mechlorethamine C-C interstrand crosslink formed is dependent on the GC:AT content of the base pairs flanking the C-C mismatch.........................................................................44 2.3.4 The electrophoretic mobility of the mechlorethamine C-C crosslinked species is dependent on the GC:AT content of the base pairs flanking the C-C mismatch............................................48 2.3.5 Sequential replacement of A-T pairs by G-C pairs has a predictable effect on the amount of C-C crosslinked DNA and on the electrophoretic mobility of the crosslink................................48 2.3.6 Molecular dynamics simulations suggest the C-C mismatched pair is stacked more favorably within a d[GCC]*d[GCC] sequence than in a d[ACT]»d[ACT] sequence..........................................51 2.4 Discussion............................................................................................................. 54 Chapter III: DNA Interstrand Crosslink Formation by Mechlorethamine at a Cytosine-Cytosine Mismatched Pair: Electrophoretic Mobility of the Crosslinked Duplexes................. 57 3.1 Introduction........................................................................................................... 57 3.2 Materials and Methods......................................................................................... 59 3.3 Results................................................................................................................... 61 3.3.1 The mechlorethamine C-C crosslink forms in DNA duplexes with single or multiple C-C mismatched pairs.............................................61 3.3.2 The mechlorethamine C-C crosslinked DNA duplexes have variable mobility on a denaturing polyacrylamide gel................................65 3.3.3 DNA duplexes having an identical mechlorethamine C-C crosslink can have variable DP AGE mobility as a function of the position of the 5'-32P-phosphate label...................................................66 3.3.4 The DP AGE mobility of the mechlorethamine C-C crosslinked duplex depends on the position of the crosslink in the duplex, and on the location of the 5'-32P-phosphate label..............67 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. vi 3.3.5 The influence of crosslink position on mobility is unchanged in duplexes carrying a 5 '-fluorescein phosphoramidite label, but the influence of the label position on mobility does change...............71 3.3.6 Duplexes carrying both 5 '-fluorescein phosphoramidite and 5'- P-phosphate labels show only small differences in duplex mobility as a function of label position........................................................ 72 3.4 Discussion.............................................................................................................. 76 Chapter IV: Extrahelical Cytosine Bases in DNA Duplexes Containing d[GCC]n «d[GCC]n Repeats: Detection by a Mechlorethamine Crosslinking Reaction......................................... 81 4.1 Introduction........................................................................................................... 81 4.2 Materials and Methods.................................................................................... 86 4.3 Results....................................................................................................................88 4.3.1 A DNA duplex containing two C-C mismatched pairs gives four crosslinked species with mechlorethamine....................................... 88 4.3.2 Extrahelical cytosine bases can be crosslinked by mechlorethamine: Evidence for an extended E-motif DNA (eE-DNA) conformation........ 92 4.3.3 A kinetic analysis suggests that multiple mechlorethamine C-C crosslink formation can occur in a single duplex.............................93 4.3.4 A duplex containing three C-C mismatched pairs gives three intrahelical and two extrahelical C-C crosslinked species with mechlorethamine... 96 4.3.5 Double crosslinks can also form in a duplex containing three C-C mismatched pairs.................................................................................99 4.3.6 A duplex containing two contiguous d[CCG]»d[CCG] repeats does not undergo crosslinking of cytosine bases that are not formally paired... 101 4.3.7 Quantification of the mechlorethamine C-C crosslinking reactions.... 101 4.4 Discussion............................................................................................................104 Chapter V: Polyamine Inhibition of Mechlorethamine Cytosine-Cytosine Crosslinking Reaction with a DNA Duplex Containing a d[GCC]2-d[GCC]2 Fragment..............................................................107 5.1 Introduction......................................................................................................... 107 5.2 Materials and Methods....................................................................................... 112 5.3 Results..................................................................................................................114 5.3.1 The mechlorethamine C-C crosslinks are inhibited by spermine..........................................................................114 5.3.2 The spermine concentrations required to inhibit mechlorethamine crosslinking at intrahelical and extrahelical cytosine mismatched bases are similar.........................................................................................115 5.4 Discussion............................................................................................................118 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Chapter VI: Effects of Cytosine Methylation on Structures of DNA Duplexes Containing a d[GCC]2«d[GCC]2 Fragment Detected by a Mechlorethamine Crosslink Reaction.............................................121 6.1 Introduction......................................................................................................... 121 6.2 Materials and Methods....................................................................................... 123 6.3 Results..................................................................................................................126 6.3.1 A duplex containing two C-C mismatched pairs within a d[GCCGCC]®d[GCCGCC] fragment gives four crosslinked species with mechlorethamine.................................................................. 126 6.3.2 A duplex containing two C-CM e mismatched pairs within a d[GCCGCC]»d[GCM e CGCM e C] fragment gives five crosslinked species with mechlorethamine.................................................................. 126 6.3.3 A duplex containing two CM e -C mismatched pairs within a d[GcM e CGCM e C]»d[GCCGCC] fragment gives five crosslinked species with mechlorethamine.................................................................. 128 6.3.4 A duplex containing two N-N mismatched pairs within a d[GCM e CGCM e C].d[GCM e CGCM e C] fragment gives six crosslinked species with mechlorethamine..................................................................128 6.3.5 A duplex containing two C-C mismatched pairs within a d[GCCGCC]»[GCCM e GCCM e ] fragment gives five crosslinked species with mechlorethamine.................................................................. 129 6.3.6 A duplex containing two C-C mismatched pairs within a d[GCCM e GCCM e ] »d[GCCGCC] fragment gives four crosslinked species with mechlorethamine.................................................................. 130 6.3.7 A duplex containing two C-C mismatched pairs within a d[GCCM e GCCM e ] «d[GCCM e GCCM e] fragment gives six crosslinked species with mechlorethamine..................................................................131 6.3.8 Quantification of the mechlorethamine crosslinked duplexes...............131 6.4 Discussion............................................................................................................133 Chapter VII: Mechlorethamine Crosslinking Reaction with a Hairpin Formed by a d[GCC]s Single strand............................................. 135 7.1 Introduction......................................................................................................... 135 7.2 Materials and Methods....................................................................................... 136 7.3 Results..................................................................................................................139 7.3.1 A dfGCCJs undergoes the mechlorethamine crosslinking reaction.....................................................................................139 7.4 Discussion............................................................................................................139 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Chapter VIII: High Performance Liquid Chromatography Determination of Mechlorethamine Crosslinking with DNA Duplexes Containing a C-C Mismatched Pair.......................................... 143 8.1 Introduction......................................................................................................... 143 8.2 Materials and Methods..................................................................................... 145 8.3 Results..................................................................................................................147 8.3.1 Synthesis and purification of mechlorethamine crosslink of DNA duplex 1........................................................................................... 147 8.3.2 Synthesis and purification of mechlorethamine crosslink of DNA duplex II..........................................................................................147 8.4 Discussion............................................................................................................148 Chapter IX: Discussion............................................................................................. 155 9.1 Summary..............................................................................................................155 9.2 Biological Significance of an eE-DNA conformation...................................... 164 References....................................................................................................................166 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. ix LIST OF TABLES Table 1.1. Characteristics of Triplet Repeat Expansion Diseases (TREDs) Type I...2 Table 1.2. Characteristics of Triplet Repeat Expansion Diseases (TREDs) Type II..3 Table 1.3. Summary of Alternative Structures formed by d[CTG]n «d[CAG]n , d[CGG]n.d[CCG]„, and d[GAA]n-d[TTC]n................................................6 Table 2.1. Kinetic parameters for mechlorethamine crosslinking of duplexes containing a C-C mismatch crosslink site and a 1,3 G-G crosslink site..42 Table 2.2. Mechlorethamine C-C mismatch crosslink formation, electrophoretic mobility of the crosslinked duplex, and duplex melting temperatures (Tm )...................................................... 47 Table 2.3. Mechlorethamine C-C mismatch crosslink formation and electrophoretic mobility of the crosslinked duplex..................................49 Table 3.1. Electrophoretic mobility of duplexes containing single and multiple C-C mismatch pairs and single mechlorethamine C-C crosslinks 62 Table 3.2. DNA duplexes containing single C-C mismatch pairs.............................69 Table 4.1. Quantification of mechlorethamine crosslinking of duplexes containing intrahelical and extrahelical C-C crosslink sites..................103 Table 5.1. Spermine inhibition of mechlorethamine crosslinking of duplex d[CTCTCGCCGCCGCCGTATC]. d[GATACGGCGCCGCCGAGAG]........................................................116 Table 5.2. Comparison of spermine inhibition of mechlorethamine crosslinking between intrahelical and extrahelical C-C crosslinking sites of duplex d[CTCTCGCCGCCGCCGTATC]. d[GATACGGCGCCGCCGAGAG]........................................................117 Table 6.1. Quantification of mechlorethamine crosslinking of duplexes I-VII.... 132 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. X LIST OF FIGURES Figure 1.1. Schematic representation of possible hairpins formed by single stranded molecules A. d[CTG]n, B. d[CAG]n, C. d[CCG]n, andD. d[CGG]n........................................................................................... 8 Figure 1.2. An intramolecular triplex DNA structure formed by d[GAA]n«d[TTC]n......................................................................................10 Figure 1.3. A sticky DNA structure formed by two intramolecular triplexes of d[GAA]n.d[TTC]n ..................................................................................... 10 Figure 1.4. Two possible alignments of quadruplex DNA structures formed by d(GG[CGG]n )and d[GGC]„.................................................... 11 Figure 1.5. Slipped-strand DNA structures (S-DNA)................................................ 12 Figure 1.6. Replication slippage model....................................................................... 15 Figure 1.7. Possible mechanisms leading to the formation of secondary structures during DNA replication.............................................................................17 Figure 1.8. Possible mechanism for massive expansions of triplet repeats during DNA replication.............................................................................19 Figure 1.9. Mechanism of triplet repeat expansions resulted from DNA repair......20 Figure 1.10. Strand breaking mechanism of expansion............................................. 21 Figure 1.11. Recombinational repair (gene conversion) model for the expansion of d[CTG]n»d[CAG]n repeat sequences.................................................23 Figure 1.12. Schematic representation of the possible conformers of a single strand of d[CCG]n.........................................................................29 Figure 1.13. Mechlorethamine and representations of DNA interstrand crosslinks induced by mechlorethamine................................................31 Figure 2.1. Schematic representation of two possible alignments of d[CCG]n 36 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. xi Figure 2.2. Mechlorethamine and representations of DNA interstrand crosslinks induced by mechlorethamine.................................................. 36 Figure 2.3. Kinetics of mechlorethamine DNA interstrand crosslink formation for reaction times up to 120 minutes....................................................... 43 Figure 2.4. Kinetics of the mechlorethamine DNA interstrand crosslink formation for reaction times up to 24 hours............................................................. 49 Figure 2.5. Autoradiogram of a 20% DP AGE gel following incubation for 6 hours with 100pM mechlorethamine of duplexes Ilia to Ille (Table III).......50 Figure 2.6. Data from molecular dynamics simulations of duplexes Ila' and IIj'...52 Figure 2.7. Structures taken after 60ps of the molecular dynamics simulations of duplex Ila' and duplex Ilj'....................................................................53 Figure 3.1. Mechlorethamine and a representation of the DNA interstrand crosslink induced by mechlorethamine at a C-C mismatch pair........................... 58 Figure 3.2. Autoradiogram of a 20% DP AGE gel showing the products of incubation of duplexes of sequence shown in Table 3.1 with 100pM mechlorethamine or no mechlorethamine................................. 63 Figure 3.3. Autoradiograms of 20% DP AGE gels following incubation 'I'j with 1 OOpM mechlorethamine of 5 '-end P-labeled duplexes IVa, IVb, IVc, Va and Vb (Table 3.2)..................................................... 70 Figure 3.4. Autoradiogram of a 20% DP AGE gel following incubation with lOOpM mechlorethamine of 5 '-fluorescein phosphoramidite-labeled duplexes IVa, IVb and IVc (Table 3.2).................................................... 73 Figure 3.5. Autoradiograms of 20% DP AGE gels following incubation with lOOpM mechlorethamine of 5 '-fluorescein phosphoramidite-labeled duplexes VI and VII (Table 3.2)..............................................................74 Figure 3.6. Autoradiogram of a 20% DP AGE gel following incubation with lOOpM mechlorethamine of duplexes IVa, IVb and IVc (Table 3.2) carrying both 5'-3 2 P-phosphate and 5'-fluorescein phosphoramidite labels........75 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. xii Figure 4.1. Schematic representation of the possible conformers of a d[GCC]3 »d[GCC] 3 duplex fragment......................................................... 84 Figure 4.2. A. Mechlorethamine. B. A representation of the DNA interstrand crosslink formed by mechlorethamine at a C-C mismatch pair and C. Duplex sequences containing C-C mismatch pairs.....................85 Figure 4.3. Autoradiogram of a 20% DP AGE gel showing the products of incubation for 1 hour of mechlorethamine with duplex II..................... 90 Figure 4.4. Possible mechlorethamine-crosslinked species for duplex II................ 91 Figure 4.5. Kinetics of mechlorethamine interstrand crosslink formation with duplex II (Figure 4.2C).....................................................................94 Figure 4.6. Kinetics of mechlorethamine interstrand crosslink formation with duplex I (Figure 4.2C)...................................................................... 95 Figure 4.7. Autoradiogram of a 20% DP AGE gel showing the products of Incubation for 1 hour of mechlorethamine with duplex III...................97 Figure 4.8. Mechlorethamine-crosslinked species for duplex III (Figure 4.2C)......98 Figure 4.9. Kinetics of mechlorethamine interstrand crosslink formation with duplex III (Figure 4.2C) for reaction times up to 4 hours...........100 Figure 4.10. Kinetics of mechlorethamine interstrand crosslink formation with duplex IV (Figure 4.2C) for reaction times up to 6 hours 102 Figure 5.1. Schematic representation of the possible conformers of d[CCG]n .... 108 Figure 5.2. Structures of polyamines. A. Spermine B. Spermidine C. Putrescine............................................................................................109 Figure 5.3. Mechlorethamine-crosslinked species for duplex d[CTCTCGCCGCCGCCGTATC]. d[GATACGGCGCCGCCGAGAG]...................................................... I l l Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. xiii Figure 5.4. Autoradiogram of a 20% DP AGE gel showing spermine inhibition of mechlorethamine interstrand crosslinks for duplex d[CTCTCGCCGCCGCCGTATC]. d[G AT ACGGCGCCGCCGAGAG]...................................................... 115 Figure 5.5. Spermine inhibition of mechlorethamine crosslinking of duplex d[CTCTCGCCGCCGCCGTATC]. d[GATACGGCGCCGCCGAGAG]...................................................... 116 Figure 5.6. Comparison of spermine inhibition of mechlorethamine C-C crosslinking of duplex d[CTCTCGCCGCCGCCGTATC]. d[GAT ACGGCGCCGCCGAGAG]...................................................... 117 Figure 6.1. Mechlorethamine-crosslinked species for d[CTCTCGCCGCCGC-CGTATC]. d[GAT ACGGCGCCGCCGAGAG]...................................................... 122 Figure 6.2. Duplexes I-VII containing two mismatched base pairs at positions 7-32 and 10-32.....................................................................123 Figure 6.3. Autoradiogram of a 20% DP AGE gel showing the products of the incubation of mechlorethamine with duplexes I-VII (Figure 6.2).......127 Figure 7.1. Autoradiogram of a 20% DP AGE gel with different exposure time (A and B), showing the mechlorethamine crosslinking of a hairpin formed by a single-stranded d[GCC] 5 ................................................... 140 Figure 7.2. Schematic representation of two possible conformers of a d[GCC]5 hairpin...............................................................................141 Figure 8.1. Mechlorethamine and representation of DNA interstrand crosslinks induced by mechlorethamine at a C-C mismatch pair......................... 144 Figure 8.2. Duplexes containing a C-C mismatch pair............................................ 145 Figure 8.3. HPLC of parent and crosslinked DNA of duplex 1...............................149 Figure 8.4. HPLC of parent and crosslinked DNA of duplex II..............................150 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. ABBREVIATIONS CM e 5 -Methylcytosine DMS Dimethyl Sulfate DMSO Dimethyl Sulfoxide DP AGE Denaturing Polyacrylamide Gel Electrophoresis eE-DNA An Extended E-Motif DNA FMR1 gene Fragile X Mental Retardation 1 gene FMRP Fragile X Mental Retardation Protein M.S. Mass Spectrometry N.M.R. Nuclear Magnetic Resonance PAGE Polyacrylamide Gel Electrophoresis S-DNA Slipped DNA SI-DNA Slipped Intermediate DNA TRE Triplet Repeat Expansion TREDs Triplet Repeat Expansion Diseases Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. XV ABSTRACT The cytosine-cytosine (C-C) pair is one of the least stable DNA mismatched pairs. The bases of the C-C mismatch are only weakly hydrogen bonded. Due to the flexibility of the mismatched cytosine bases, DNA containing C-C mismatched pairs has the potential to adopt a variety of unusual DNA conformations. The goal of this dissertation is to identify the structures of C-C mismatched pairs within DNA triplet repeat sequences using a mechlorethamine crosslinking reaction at cytosine-cytosine bases as a probing reaction. Here, using DNA duplexes with d[GCC]n ®d[GCC]n repeat fragments containing C-C mismatches in a 1,4 base pairs relationship, it is shown that mechlorethamine can crosslink at all formal C-C mismatched pairs, and between two mismatched cytosine bases that are not formally paired (1,4 C-C bases). It has been shown previously that the bases of a single C-C mismatch within a d[GCC]*d[GCC] fragment can become unstacked from the core helix, and adopt an ‘extrahelical’ location (in the so-called E-motif conformation). Hence, the formation of the 1,4 C-C crosslinks is interpreted as crosslinking at extrahelical cytosines. In the E-motif, the extrahelical cytosines are folded back towards the 5' end of the duplex, consistent with the formation of 1,4 C-C crosslinks and the absence of 4,1 C- C crosslinks in the current work. The data provide evidence for an extended E-motif DNA (eE-DNA) conformation in d[GCC]n*d[GCC]„ repeat fragments. Since the d[GCC]n»d[GCC]n repeat fragment resembles the stem of intramolecular hairpins Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. xvi formed by single-stranded d[CCG]n, the eE-DNA conformation may be involved in an expansion and hypermethylation of d[CGG]n»d[CCG]n triplet repeats associated with Fragile X syndrome. Hence, the discovery of the novel eE-DNA conformation may contribute to an understanding of the disease development. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 1 CHAPTER I Introduction 1.1 Diseases Associated with DNA Triplet Repeat Expansion The human genome contains several kinds of repeated DNA sequences, such as inverted repeats, mirror repeats, and runs of a single nucleotide and di-, tri- or higher-order nucleotide repeats (Charlesworth et al., 1994). These repeated DNA sequences are hot spots that frequently undergo spontaneous mutagenesis (Sinden, 1999). In 1991, a novel mutation type associated with an increased number of trinucleotide (triplet) repeats in the human genome was discovered (Kremer et al., 1991; Ververk et al., 1991; Yu et al., 1991,1992). This mutation is called a triplet repeat expansion (TRE). Subsequently, it has been found that TRE is linked to several neurological and neuromuscular disorders, the so-called triplet repeat expansion diseases (TREDs) (for reviews see Sutherland and Richards, 1995a,1995b; Pearson and Sinden, 1998; Timchenko and Caskey, 1999; Bowater and Wells, 2001). These diseases can be divided into two categories based on the characteristics of expansions of triplet repeat sequences, namely type I and type II (for reviews, see Paulson and Fischbeck, 1996; Reddy and Housman, 1997). The characteristics of TREDs are summarized in Table 1.1 and Table 1.2. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Reproduced w ith permission o f th e copyright owner. Further reproduction prohibited without permission. Table 1.1. Characteristics of Triplet Repeat Expansion Diseases (TREDs) Type I Disease* (Type I) Inheritance Gene/ Locus Chromosomal Localization Protein Product Repeat Sequence Normal Length Pre mutation Length Mutation Length Inter ruption Repeat Location SMBA (Kennedy’s disease) Recessive AR Xql3-21 Androgen Receptor [CAGJ«[CTG] 11-13 none 38-66 none Coding Huntington’s disease Dominant IT15 4pl6.3 Huntingtin [CAG]«[CTG] 3-39 none 36-121 none Coding DRPLA (HRS) Dominant DRPLA (B37) I2pl 3.31 Atrophin-1 (DRPLAP) [CAG]»[CTG] 6-35 none 51-88 none Coding SCA type 1 Dominant SCA1 6p23 Ataxin-1 [CAG]»[CTG] 6-39 none 41-81 CAT Coding SCA type 2 Dominant SCA2 12q24.1 Ataxin-2 [CAG].[CTG] 14-31 none 35-64 CAA Coding SCA type 3 (MJD) Dominant SCA3 (MJD1) 14q32.1 Ataxin-3 |CAG].|CTG] 12-41 none 40-84 none Coding SCA type 6 (EA2) Dominant CACNA1A 19pl3 x 1 A-voltage dependent calcium channel subunit [CAG]»[CTG] 7-18 none 20-23 (EA2) 21-27 (SCA6) none Coding SCA type 7 Dominant SCA7 3pl2-13 Ataxin-7 [CAG]o[CTG] 7-17 none 38-130 none Coding Abbreviations: SBMA, Spinobulbar atrophy; DRPLA, Dentatorubral-pallidoluysian atrop HRS, Haw river syndrome 11 y; This table was modified from Bowater and Wells, 2001. t o Reproduced w ith permission o f th e copyright owner. Further reproduction prohibited without permission. Table 1.2. Characteristics of Triplet Repeat Expansion Diseases (TREDs) Type II Disease (Type II) Inheritance Gene/ Locus Chromosomal Localization Protein Product Repeat Sequence Normal Length Pre mutation Length Mutation Length Inter ruption Repeat Location Fragile X syndrome Dominant FMR1 (FRAXA) Xq27.3 FMRP [CGG]«[CCG] 6-52 60-200 230-1000 AGG 5-U TR Fragile XE mental Retardation Dominant? FMR2 (FRAXE) Xq28 FMR2 [GCC]»[CGG] 7-35 130-150 230-750 none 5'-UTR Jacobsen Syndrome Dominant FRA11B 1 lq23 CBL2 Proto oncogene [CGGMCCG] 11 80 100-1000 none 5’-UTR Friedreich’s ataxia Recessive FRDA/ X25 9ql3-21.1 Frataxin [GAA]«[TTC] 6-34 34-40 112-1700 GAGG AA Intron 1 Myotonic Dystrophy Dominant DMPK 19ql3 Myotonic Dystrophy protein kinase [CTG]»[CAG] 5-37 50-80 80-3000 none 3 '-UTR This table was modified from Bo water and Wells, 2001. U> 4 1.2 Triplet Repeat Expansion Diseases Type I The type I diseases are characterized by small expansions (of the order of tens of repeats) of d[CAG]n «d[CTG]n triplet repeats within the coding regions of various genes, resulting in the synthesis of longer tracts of polyglutamines in the relevant proteins (Table 1.1). The diseases exhibit neurological problems associated with neuronal loss in the brain, brain stem and spinal cord (Robitaille et al, 1997; Timchenlco and Caskey, 1999). Examples of type I TREDs are Huntingtion’s disease (HD), spinal and bulbar muscular atrophy (SBMA) or Kennedy’s disease, dentatorubral-pallidoluysian atrophy (DRPLA) or Haw River syndrome, Machado- Joseph disease or spinocerebella ataxia type 3 (MJD/SCA3), and spinocerebellar ataxias types 1, 2, 6, and 7 (Bowater and Wells, 2001). 1.3 Triplet Repeat Expansion Diseases Type II The characteristics of type II TREDs involve large expansions (of the order of hundreds or even thousands of repeats) of different sequences of triplet repeats (d[CTG]n«d[CAG]n , d[CGG]„.d[CCG]„, and d[GAA]n»d[TTC]n ), which are located in various regions of their affected genes (Table 1.2). Another unique characteristic is that there is an intermediate length of triplet repeats found in carriers of the type II diseases. In addition, the type II diseases not only display neurological problems, but also a complexity of symptoms in many tissues. Examples of type II TREDs are Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 5 Fragile X syndrome (FRAXA), Fragile XE mental retardation (FRAXE), Jacobsen syndrome (FRA11B), Friedreich’s ataxia (FRDA), and myotonic dystrophy (MD) (Mahadevan et al., 1992; Tapscott et al., 1998; Thornton, 1999; Bowater and Wells, 2001). For more details of the pathology of the TREDs, the reader is referred to the following reviews: (Paulson and Fischbeck, 1996; Timchenko and Caskey, 1996; Harris et al., 1996; Reddy and Housman et al., 1997; Price et al., 1998; Martin, 1999). An inclusive review of Fragile X syndrome is included in section 1.6. 1.4 Alternative DNA Structures Formed in Triplet Repeat Sequences Triplet repeat expansion has been linked to alternative structures formed by triplet repeat sequences associated with TREDs (Mitas, 1997; Pearson and Sinden, 1998; Sinden, 1999). Because of this, the conformational properties of d[CGG]n» d[CCG]n, d[CTG]n«d[CAG]n , and d[GAA]n»d[TTC]n have received considerable attention. The alternative structures formed by these triplet repeats can be hairpins, triplexes, quadruplexes, and slipped-strand DNA, depending on the repeat sequences (Table 1.3) (for reviews see Wells, 1996; Mitas, 1997; Gellibolian et al., 1997; Gacy and McMurray, 1998; Pearson and Sinden, 1998; Lunkes et a l, 1998; Darlow and Leach, 1998a; Sinden, 1999; Bowater and Wells, 2001). The details of these structures are described below. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 6 Table 1.3. Summary of Alternative Structures Formed by d[CTG]n»d[CAG]n, d[CGG]n.d[CCG]n, and d[GAA]n»d[TTC]n Triplet Repeat Sequence Alternative Structures d[CTG]n«d[CAG]n Slipped DNA (S-DNA) Slipped intermediate DNA (SI-DNA) d[CTG]n Intrastrand hairpins Interstrand duplexes d[CAG]n Intrastrand hairpins d[CGG]n»d[CCG]n Slipped DNA (S-DNA) Slipped intermediate DNA (SI-DNA) d[CGG]n Intrastrand hairpins Interstrand duplexes Quadruplex DNA d[CCG]n Intrastrand hairpins Interstrand duplexes d[GAA]n»d[TTC]n Intramolecular triplex DNA 1.4.1 Unusual Characteristics of DNA Duplexes Containing Triplet Repeats DNA duplexes containing d[CTG]n»d[CAG]n or d[CGG]n«d[CCG]n triplet repeats have shown many unusual characteristics that have been detected with biochemical analysis and electron microscopy. These duplexes exhibited higher flexibility and faster electrophoretic mobility in polyacrylamide gels than random B- DNA duplexes (Chastain et al., 1995; Chastain and Sinden, 1998). These unusual properties, which differ from those of mixed sequence DNA, indicate that Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 7 d[CTG]n«d[CAG]n and d[CGG]n «d[CCG]n repeats are flexible and writhed (Bacolla et al., 1997; Chastain and Sinden, 1998). However, both repeats showed an opposite behavior with respect to nucleosome assembly. d[CTG]„»d[CAG]n repeats bound to nucleosomal proteins with the highest efficiency (Wang et al., 1994; Wang and Griffith, 1995,1996a, 1996b; Godde and Wolffe, 1996) whereas d[CGG]n »d[CCG]n repeats bound to nucleosomal proteins with the least efficiency (Godde et al., 1996). For d[CGG]n»d[CCG]n , the ability to bind to the nucleosomal protein can be affected by methylation. The methylation of d[CGG]n»d[CCG]n repeats with a normal length (n=13) increases the binding efficiency, but the effect of methylation disappears in repeats with a pre-mutation length (n = 74-76) (Wang and Griffith, 1995; Godde et al., 1996). These results suggest that a difference in chromatin organization might depend on the state of repeat expansion and methylation (Pearson and Sinden, 1998). 1.4.2 Intramolecular Hairpin Structures in Single-Stranded d[CTG]n , d[CAG]n , d[CGG]n , and d[CCG]n The single-stranded DNA molecules of d[CTG]n, d[CAG]n , d[CGG]n, and d[CCG]n can adopt intrastrand hairpin conformations, as shown in Figure 1.1 (Darlow and Leach, 1998a, 1998b; Pearson and Sinden, 1998). These hairpins contain a mismatched pair (T-T, A-A, G-G, or C-C) and two Watson-Crick pairs (C- G and G-C) within every three base pairs of triplet repeats. Single-stranded d[CTG]n and d[CAG]n can each form a hairpin structure containing a T-T or an A-A Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 8 mismatched pair (Figure 1.1 A and B) (Yu et al., 1995a,1995b; Mitas et al., 1995a; Petruska, 1996; Mariappan, 1996a). In contrast, both single-stranded d[CCG]n and d[CGG]n can each form hairpin structures with two different alignments (Figure 1.1C and D). These alignments contain either a d[GC]*d[GC] or a d[CG]«d[CG] Watson-Crick dinucleotide step with either a C-C or a G-G mismatched pair (Nadel et al., 1995; Mariappan et al., 1995, Mitchell at al., 1995; Mitas et al., 1995b; Chen et al., 1995; Zheng et al., 1996; Yu et al., 1997). For d[CGG]n repeat, the hairpin with a d[CG]«d[CG] dinucleotide step is the preferred conformation (Figure l.lD(right)) (Chen et al., 1995; Mitas, 1995b; Zheng et al., 1996). For small repeats of d[CCG]n (n = 5-7), the hairpin with a d[GC]»d[GC] dinucleotide step is the preferred conformation (Figure l.lC(left)) (Chen et al., 1995; Zheng et al., 1996; Mariappan et al., 1996b). However, for the longer repeats (n = 15 or more), d[CCG]n formed distorted hairpins with a d[CG]«d[CG] dinucleotide step (Figure l.lC(right)) (Yu etal., 1997). A B C D r\ r\ G • C G • C T o T A o A C • G C • G I I G • C G * C G * C T o T A o A C o C C • G C • G C • G G • C G • C G • C T o T A o A C o C C • G C * G C • G 5' 5' 5' Figure 1.1. Schematic representation of possible hairpins formed by single-stranded A. d[CTG]n, B. d[CAG]n, C. d[CCG]n, and D. d[CGG]n. For C. and D., left and right panels represent d[GC>[GC] and d[CG]«[CG] Watson-Click dinucleotide steps, respectively. A filled bullet (•) and a hollow bullet (o) indicate Watson-Crick pairs and mismatched base pairs, respectively. n r \ C • G C o C n C • G G o G G • C G • C G * C C • G G o G C • G C o C C • G G o G G • C G • C G • C C * G G o G C • G C C • G G 5' 5' 5' Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 9 1.4.3 Triplex DNA Structures in d[GAA]n «d[TTC]n It has long been proposed that triplex DNA formation is involved in repeat expansions of d[GAA]n»d[TTC]n repeats in Friedreich’s ataxia. Intramolecular triplex structures have been shown to block DNA replication in vitro and in vivo (Rao et al., 1988; Krasilnikov et al., 1997). An intramolecular triplex structure of d[GAA]n »d[TTC]n repeats is shown in Figure 1.2. High-resolution structures of the triplex formed by d[GAA]n*d[TTC]n have been characterized using nuclear magnetic resonance (NMR) techniques. Mariappan et al. (1999) have found that the N.M.R. structures of two intramoleculary folded triplexes, [GAA]2 T4 [TTC]2 T4 [CTT] 2 and [GAA]2 T4[TTC]2 T2 CT2 [CTT] 2 contain T-A.T and C+ .G«C triads and the cytosine residues of the Hoogsteen C+ »G pairs in the triplex are protonated close to the physiological pH (Mariappan et al., 1999). Recently, Sakamoto et al. (1999) discovered a novel structure, termed “sticky DNA” (Figure 1.3). This structure was formed by the association of two purine»purine*pyrimidine triplexes in negatively supercoiled plasmid at neutral pH (Sakamoto et al., 1999). Hence, the formation of triplex and sticky DNA offers a plausible explaination for the expansion of d[GAA]n»d[TTC]n repeats in Friedreich’s ataxia. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 10 G A A GAA'O A A & A AQ A O, I T C & 0 0 ~ A A G • * • <5 A T r C T I C TT - - . j 490 Q99 i}p^C ,T T T C 3 & 0 . , . _ r _ _ . AA0 AAQ A A <3 A A * * • * • TT C T T C T T C T T C TT •C-T Figure 1.2. An intramolecular triplex DNA structure formed by d[GAA]n«d[TTC]n. A filled bullet (•) indicates Watson-Crick pairs and a hollow bullet (o) indicates Hoogsteen base pairs (Pearson and Sinden, 1998). am 'CTTCTT ! 1 i t I I f l i l l l l l f f GAAGAAGAAGAAGAAGAA-'v ***********;**-***** J g a a g a a g a a g a a g a a o a a ^ ***** *'*•** . S&AGRASMUSAAOAXaAA I i l t l M I M / I U inn ■ C T T C M C 5 Figure 1.3. A sticky DNA structure formed by two intramolecular triplexes of d[GAA]n»d[TTC]n. A short vertical line (|) between the bases indicates Watson- Crick pairs and a star (★) indicates reversed Hoogsteen base pairs (Sakamoto et al., 1999). Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 11 1.4.4 Quadruplex DNA Structures in Single-Stranded d[CGG]n It has been reported that single-stranded d[CGG]n can form quadruplex DNA structures held together by G «G Hoogsteen hydrogen bonding and possibly by C® C hydrogen bonds (Fry and Loeb, 1994; Usdin and Woodford, 1995; Kettani et al, 1995; Usdin, 1998). Two possible alignments of quadruplex DNA structures have been suggested, as shown in Figure 1.4 (Usdin 1998). The formation of quadruplex structures requires the presence of cations, with the order of stabilizing ability for cation as: K+ > Ca2 + > Na+ > Mg2 + (Hardin et al., 1993). Usdin and Woodford (1995) have demonstrated that specific pauses in DNA replication required the presence of K+ ions and the d[CGG]n template strand. This condition is also required for the formation of quadruplex structures. They have interpreted this correlation to mean that the pause in replication may be due to the quadruplex formation. Therefore, it is possible that quadruplex formation may be involved in the etiology of Fragile X syndrome. Figure 1.4. Two possible alignments of quadruplex DNA structures formed by d(GG[CGG]n) and d[GGC]n (Pearson and Sinden, 1998). Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 12 1.4.5 Slipped-Strand DNA Structures Long triplet repeat sequences can form slipped-strand DNA structures, as first described by Sinden and co-workers (Pearson and Sinden, 1996). These structures are caused by an out-of-register misalignment of complementary duplex strands resulting in the formation of loops and unpaired DNA within each strand (Figure 1.5). There are two types of slipped-stranded DNA structures, which are homoduplex slipped DNA (S-DNA) structures (Pearson and Sinden, 1996) and heteroduplex slipped intermediates DNA (SI-DNA) structures (Pearson el al., 1997). S-DNA can be formed between complementary strands of triplet repeats with the same number of repeats (Pearson and Sinden, 1998). In contrast, SI-DNA can be formed between strands of triplet repeats with a different number of repeats (Pearson and Sinden, 1998). Figure 1.5. Slipped-strand DNA structures (Pearson and Sinden, 1998). A. Slipped intermediate structures (SI-DNA) formed by the slippage of (i) the nascent strand or (ii) the template strand. B. Various slipped structures (S-DNA) formed following denaturation and renaturation of a triplet repeat tract in an out of register fashion (i- iv). Thin lines represent repeat tracts, and broad lines are non-repetitive flanks. A B < 9 mat... fi .n n..n.— ^ X J U L A w Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 13 The complexity and the number of S-DNA structures are dependent on the length of the repeats and the presence of interrupted sequences within the repeats (Pearson and Sinden, 1998). The complexity and the number of S-DNA structures increase with increasing length of the repeat tracts. In normal individuals, the triplet repeats contain interrupted sequences (as listed in Table 1.1 and 1.2) for repeat tract stability. The loss of the sequence interruption results in a longer length of the pure tract and hence increases the complexity and the amount of S-DNA isomers. Therefore, the effects of the length and of the sequence interruptions on the propensity of S-DNA formation correlate with their effects on genetic stability, expansion and disease (Pearson and Sinden, 1998). 1.5 Molecular Mechanism of Triplet Repeat Expansion/Deletion Although it has been known since 1991 that several neurological diseases are associated with large expansions of triplet repeats within the human genome, the molecular mechanism of triplet repeat expansion is still unknown. A wide variety of experiments have been established in E. coli, yeast, transgenic mice, mammalian cell culture models, and in human clinical cases to investigate the expansion (and also deletion) mechanisms of triplet repeats (for a review, see in Bowater and Wells, 2001). These expansions and deletions are believed to be due to the formation of unusual secondary structures and slipped complementary strands during DNA biochemical processes including replication (Kang et al., 1995a,1995b,1996; Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 14 Ohshima et al., 1996; Ohshima and Wells, 1997; Samadashwily et al., 1997; Petruska et a l, 1998; Darden-Lyons and Topal; 1999), repair (Jaworski et al, 1995; Pamiewski et al., 1999; Wells et al., 1998; Schumacher et al, 1998; Richard et al., 1999; Darden-Lyons and Topal; 1999), and recombination (Richardson et al., 1998; Jakupciak and Wells, 1999; Jakupciak and Wells, 2000a,2000b). During these pathways, the DNA complementary strands are separated and each strand has the potential to rearrange itself and adopt unusual DNA structures which could lead to repeat expansions. The stability of the repeat tracts is dependent on the ability to form unusual structures within the repeats which, in turn, relies on the repeat sequences and their length (Pearson and Sinden, 1998). Sequence interruption within the repeat tracts has been reported to stabilize the triplet repeats due to a decreased ability to form unusual structures (Pearson et al., 1998). How triplet repeat structures and DNA biochemincal processes (replication, repair and recombination) generate the genetic instability is discussed below. 1.5.1 Triplet Repeat Expansion/Deletion Caused by DNA Replication During the replication, the DNA double helix is unwound, creating the replication forks at which polymerase complexes can bind to synthesize the new DNA (Warren, 1997). If, at this time, templates or new strands form unusual DNA structures, such as hairpins (d[CTG]n, d[CAG]n, d[CGG]n and d[CCG]n), triplexes (d[GAA]n»d[TTC]n) and quadruplexes (d[CGG]n) (Figure 1.6), then expansions or Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 15 deletions events can occur. If the structures are formed within leading or lagging template strands, DNA polymerase will bypass the structures during the replication, leading to the deletions (Figure 1.6B). In contrast, if the structures are formed within the leading or lagging nascent strands, DNA polymerase activity is extended and the template is later filled in, resulting in the expansions (Figure 1.6A). Expansion Expansion product ■ * 1st round of replication 2nd round of replication Deletion SI-DNA Dfetetion product Figure 1.6. Replication slippage model showing SI-DNA dining synthesis of the triplet repeats. Slippage of the nascent strand (A.) or the template strand (B.) results in the formation of heteroduplex slipped intermediate DNA (SI-DNA). Following continued replication, the DNA contains an excess of repeats in one strand. This subsequently leads to expansion (A.) or deletion (B.) products. Thin lines represent repeat tracts, and broad lines are non-repetitive flanks (Pearson and Sinden, 1998). Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 16 Since expansions occur more frequently than deletions, it has been suggested that formation of alternative DNA structures is more likely in the lagging nascent strand (Sinden, 1999). The reasons are that (i) the lagging strand is the site where a single-stranded DNA exists during the replication, (ii) the length of this single stranded region is always equal to that of an Okazaki fragment, which occurs in a lagging nascent strand, and (iii) the normal triplet repeats in the genomes usually contain interrupted sequences and the long pure triplet repeat is no longer than the normal length of a mammalian Okazaki fragment (25-300 nucleotides) (Trinh and Sinden, 1991; Sinden et al., 1991; Kang et al., 1995; Schumacher et al., 1998; Sinden 1999). Several models explaining how unusual structures can adopt and cause expansions during DNA replication have been proposed. One model is that the perfect repeat Okazaki fragment in the lagging nascent strand is slipped during movement of polymerase (Figure 1.7A) (for reviews, see Djian, 1998; Pearson and Sinden, 1998; Bowater and Wells, 2001). The slipped-strand fragments then form hairpins or triplexes and extend the DNA synthesis of the template. The template is later filled in, resulting in the expansions. This model can explain the expansion from pre-mutation to pre-mutation variation. However, the massive expansion from pre mutation to full mutation cannot easily be explained using this DNA-slippage model (Jin and Warren, 2000). Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. PoIyCXG B PolyGAA \ N . v A c V ' Wo , ^ 4 SS FLAP i V^x4r. • . . ySy < 0 $ £ % in g strand > s t t e i | “ ► & / W ® ? r y Q i n o « r f l t w f W A I K P I M A / . ^ l n S s l l a l , ( l „< S /V in g strand HAIRPIN M /J T S e ™ # rX v^ s C N Figure 1.7. Possible mechanisms leading to the formation of secondary structures during DNA replication (Djian, 1998). (A) Slippage of Okazaki fragment in the lagging nascent strand during movement of polymerase leads to the formations either a hairpin (polyCXG) or a triplex (polyGAA). (B) A newly synthesized Okazaki fragment displaces the 5' end of the downstream Okazaki fragment, thus generating a single-stranded (SS) flap. This SS flap converts into either a hairpin or triplex flap. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 18 Another model explaining how unusual structures can adopt and cause expansions during DNA replication is the formation of single-stranded flaps at the 5' end of Okazaki fragment (Figure 1.7B) (Johnson et al., 1992; Gordenin et al., 1997; Tishkoff et al., 1997; Kokoska et al., 1998). The single-stranded flaps of of the 5' end of the preceding Okazaki fragment are generated by the displacement of the growing 3' end of the newly synthesized Okazaki fragment (Figure 1.7B). It has been known that RAD27 endonuclease in yeast and its mammalian homolog, FEN1 (5' exonuclease-1 or flap endonuclease-1), are responsible for the removal of 5' ribonucleotide, derived from the RNA primer and the 5' end of an Okazaki fragment (Goulian et a l, 1990; Lyamichev et al., 1993; Turchi and Bambara, 1993; Waga et ah, 1994). Since the FEN1 substrate must be single-stranded, formations of secondary structure within the flap may escape the endonuclease activity of FEN1 (Gacy et al., 1995; Chen et ah, 1995; Bambara et ah, 1997). This leads to the extended synthesis of the preceding Okazaki fragment, resulting in the repeat expansions. This idea is also supported by the observation that, in the absence of RAD27 or FEN-1, the triplet repeats highly increased their frequency of expansions (Freudenreich el ah, 1997; White et al., 1999). Single-stranded flaps can also be generated by strand breaking, and formation of unusual structures within these flaps can cause repeat expansions via DNA repair (Petruska et al., 1998; Sarkar et al., 1998). The details are discussed in section 1.5.2. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. For a mechanism of massive expansion, Sinden and Wells (1992) suggested that the expansion is caused by reiterative DNA synthesis occurring when DNA polymerase stalls during replication (Figure 1.8) (Sinden and Wells, 1992). Stalling sites of DNA polymerase may be caused by secondary structures forming within the triplet repeats. Once the polymerase stalls, the synthesized sequences become much longer compared to DNA templates. This model alone therefore can explain the large expansions from pre-mutation to full mutation (Sinden, 1999). B Figure 1.8. Possible mechanism for massive expansions of triplet repeats during DNA replication. A. Reiterative synthesis at a folded slipped structure in the leading strand template. B. Reiterative synthesis at a quadruplex structure in the lagging strand template (Sinden, 1999). Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 20 1.5.2 Triplet Repeat Expansion/Deletion Caused by DNA Repair Double- and single-strand breaks accompanied by DNA repair have been proposed as the cause of triplet repeat expansions (Figure 1.9) (Gordenin et al., 1997). Strand breaking within the triplet repeats creates gaps and single-stranded flaps. The flaps containing triplet repeats then form unusual structures, such as hairpins or slippage DNA. Subsequently, repair of the gap occurs, leading to repeat expansions. H X i’ A N .sii is ______.. J n111 nn immi1 1 1 m«i ini n 1 1 fi j nnn.i Figure 1.9. Mechanism of triplet repeat expansions resulting from DNA repair. A nick or gap (arrowhead) in the triplet repeat tract initiates slippage and hairpin formation. Repair of the gap leads to expansion (Djian, 1998). Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 21 Another expansion mechanism involving double-strand break and DNA repair has also been suggested (Figure 1.10) (Petruska et al., 1998; Sarkar et al., 1998). In this model, the strands of triplet repeat DNA duplexes initially slip and form hairpins within both strands, resulting in breaking of the strand opposite to the hairpins. The hairpins are then free to slide, thus generating gaps. The gaps are eventually repaired and the repeats become expanded. 5 ' Figure 1.10. Strand breaking mechanism of expansion. The strands slip and form hairpin structures that may be cleaved by nucleases. The hairpins are then free to slide and travel along the duplex and upon repair are expanded (Petruska et al., 1998; Sarkar et al., 1998) Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 22 1.5.3 Triplet Repeat Expansion/Deletion Caused by DNA Recombination DNA recombination including unequal crossing-over (Tsilfidis et al., 1992) and gene conversion (recombinational repair) (O'Hoy et al., 1993) has been proposed as the mechanism responsible for triplet repeat expansions (for a review, see Jakupciak and Wells, 2000b). Unequal crossing-over is considered an unlikely cause for triplet repeat expansion because the exchange of flanking markers was not found during the mapping of the neurological disease genes (Richards and Sutherland, 1992a, 1992b; Sutherland and Richards, 1992). Recent investigation has demonstrated that a potential mechanism for triplet repeat expansion is gene conversion (Tishkoff et al., 1997; Richardson et ah, 1998; Jakupciak and Wells, 1999). This mechanism is modeled in Figure 1.11 (for a review, see Jakupciak and Wells, 2000b). A double-strand break occurs within a triplet repeat DNA and then one strand of the DNA inserts into a gap of another double-stranded DNA. After hybridization within the gap, the recombinational repair (gene conversion) process begins to fill the nucleotide base. Ligation then occurs to complete the strands, resulting in expansions. It is possible that during the recombinational repair steps the strand may form alternative stmctures, resulting in an increased formation of expansions. Hence, these two processes work powerfully together to generate massive expansions. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 23 Figure 1.11. Recombinational repair (gene conversion) model for the expansion of d[CTG]n *d[CAG]n repeat sequences suggested by Jakupciak and Wells (2000b). Portions of the original plasmids used for the cotransformation are shown in the upper left. The heavy solid lines represent the triplet repeat sequences in either orientation I or orientation II. The medium solid lines represent the pUC19 vector sequences that flank the d[CTG]n «d[CAG]n insert. The stippled regions represent the pACYC184 vector sequences that flank the triple repeat sequences. A double-strand break occurs within the d[CTG]n»d[CAG]n tract and that is enlarged to a double strand gap (Tishkoff et al., 1997; Cordeiro-Stone et ah, 1999). Stand invasion with staggered hybridization initiates the recombinational repair process. Two Holliday- like junctions are formed and DNA repair synthesis (filled circles) takes place on both strands. Simple repeat sequences adopt slipped mispaired structures. After the formation of hairpin loop structures, further DNA synthesis may also contribute to elongation of the triplet repeat sequences. Depending on the position of the staggered hybridization, the amount of slippage and the extent of branch migration, different size expansion products will be formed. Resolution of the junctions leaves the flanking sequences unaltered (not exchanged) (Jakupciak and Wells, 2000b) Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 24 1.6 Fragile X Syndrome Fragile X syndrome, a triplet repeat expansion disease, is the most common form of inherited mental retardation with a prevalence estimated to be one in 1500 males and one in 2500 females (Warren, 1994; Ashley and Warren, 1995; Kunst, 1996; Sutherland and Richards, 1995a,1995b; Timchenko and Caskey, 1996; Warren, 1997; De Vries et al., 1998; Kaufmann and Reiss, 1999; Jin and Warren, 2000). The syndrome is transmitted as an X-linked dominant trait. The phenotype of this syndrome is mental impairment, ranging from severe retardation to low-normal intelligence, and a number of dysmorphic features: a long narrow face, large ears, prominent jaw and forehead, and macro-orchidism. Generally, the extent of phenotypic manifestation is more severe in males (Kaufmann and Reiss, 1999). Additional clinical manifestations include connective tissue abnormalities (i.e. joint hyperextensibility), autistic-like behaviors, psychiatric disorders, hyperactivity and attention deficit disorder (ADHD) (Kaufmann and Reiss, 1999). In successive generations of Fragile X patients, the age of onset is earlier and the severity increases. This phenomenon, called anticipation, is correlated to the increased lengths of triplet repeats within the genome in the next generation (Kaufmann and Reiss, 1999). Fragile X syndrome was originally identified through the fragile site at Xq27.3, termed FRAXA (FRAgile site, X chromosome, A site) (Kaufmann and Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 25 Reiss, 1999). The expression of a break on this fragile site can be induced by culturing cells in medium deficient in folic acid and thymidine, thus lowering the levels of dTTP and dCTP pools. In 1991, the gene responsible for Fragile X syndrome was identified as Fragile X Mental Retardation 1 (FMR1) gene within the fragile site at Xq27.3 (Kremer et al., 1991; Ververk et al., 1991; Yu et al., 1992). This gene spans 38 kb and is composed of 17 exons (Ashley et al., 1993a,1993b; Eichler et al., 1993). However, all the necessary elements for proper FMR1 gene expression are located in the 5 '-untranslated region consisting of the first exon and its upstream area. The exon 1 of the gene contains both the d[CGG]n»d[CCG]n triplet repeats and the start site, ATG, which is located downstream of the triplet repeats (Fu et al., 1991; Snow et al., 1993,1994; Eichler et al., 1994; Kunst et al., 1994). The promoter of this gene, the CpG island, is found in the 5' upstream region of the d[CGG]„«d[CCG]n triplet repeats. At the molecular level, Fragile X syndrome is characterized by large expansions of d[CGG]]n»d[CCG]n repeats within the 5'-untranslated region of FMR1 gene (Fu et al., 1991; Tapscott et al., 1998). In the normal population the gene contains a relatively small number of d[CGG]„»d[CCG]n repeats (Fu et al, 1991; Reiss et al., 1994; Kunst et al., 1994; Eichler et al., 1994). The repeat number is polymorphic, ranging between 6-54 with an average of 30, and the “pure” repeat sequences are often interrupted by single d[AGG]*d[CCT] triplets, restricting the Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 26 lengths of the repeats to less than 40 (Kunst et al., 1994; Eichler et al., 1994). In contrast, in Fragile X carriers the number of repeats may reach 200 (pre-mutation), and the offspring of these carriers may have thousands of repeats (full mutation) (Fu et al., 1991). The molecular consequence of repeat expansions is a failure to make an RNA transcript of the FMR1 gene, which in turn, is believed to be due to the hypermethylation of the promoter, the CpG island, of the gene (Oberle et al., 1991; Pieretti et al., 1991; Homstra et al., 1993). This phenomenon results in an absence or reduction of Fragile X Mental Retardation Protein (FMRP), which is thought to be the molecular basis for Fragile X syndrome. FMRP is believed to be an RNA-binding protein (Ashley et al., 1993b; Siomi et al., 1993). Analysis of the amino acid sequence of FMRP revealed the presence of three domains involved in RNA binding. These are two ribonucleoprotein K homology domains (KH 1 and KH 2 domains) central to the molecule and clusters of arginine and glycine residues (RGG box) near the C-terminals (Ashley et al., 1993b; Siomi et al., 1993). In 1995, two proteins produced from autosomal homologs of the FMR1 gene (FXR1 and FXR2 gene) were identified and the structures of these proteins are similar to that of FMRP (Siomi et al., 1995; Zhang et al., 1995). Due to their similarities, it has been suggested that FXR1P and FXR2P can compensate for the functions of FMRP in Fragile X patients. This leads to a non-lethal, but mild phenotype in affected individuals (Jin and Warren, 2000). Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 27 The FMR1 gene expression studies have demonstrated that FMRP is expressed in a variety of tissues including liver, lung, pancreas, kidney, and lymphocytes, with the highest level in the brain and testes (Hinds et al., 1993). Cell and tissue immunohistochemistry have shown that, within each tissue, the distribution of FMRP is cell-type specific. For example, in the testis, FMRP is mainly in spermatogonia but not so much is found in mature germ or Sertoli cells (Devys et al., 1993; Sittler et al., 1996). In the brain, FMRP is predominantly in neurons (Devys et al., 1993; Sittler et al., 1996). Studies of FMRP intracellular localization and FMRP transfections have shown that FMRP can be found in the cytoplasm, nucleoplasm and nuclear pores (Deyvs et al., 1993; Hoogeveen et al., 1997). Molecular studies also show that FMRP is a ribonucleoprotein containing nuclear localization and nuclear exporting signal (Eberhart et al., 1996). These findings suggest that FMRP could shuttle between the nucleus and cytoplasm, and the lack of FMRP may alter the transport of RNAs to which it normally binds (Deyvs et a l, 1993; Eberhart et al., 1996; Hoogeveen et a l, 1997; Timchenco and Caskey, 1999). 1.7 Dynamic Conformational Properties of Structures Formed by d[CCG]n d[CGG]n»d[CCG]„ repeat expansion (Wells, 1996; Gellibolian et al., 1997; Mitas, 1997; Pearson and Sinden, 1998; Gacy and McMurray, 1998; Sinden, 1999) and gene hypermethylation (Smith et al., 1994; Chen et a l, 1995) in Fragile X Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 28 syndrome have been linked to secondary structures formed by each strand of the d[CGG]n«d[CCG]n sequence. Because of this, the conformational properties of single-stranded d[CGG]n and d[CCG]n have received considerable attention (Fry and Loeb, 1994; Chen et al., 1995; Mitas et al., 1995b; Gacy et al., 1995; Nadel et al., 1995; Usdin and Woodford, 1995; Gacy et al., 1996; Mariappan et al., 1996b,1998; Zheng, 1996; Yu et al., 1997; Usdin, 1998; Darlow and Leach, 1998a,1998b). The dynamic conformational properties of intramolecular structures formed by d[CCG]n repeat sequences are discussed in the following text and summarized in Figure 1.12. The dynamic conformational properties of intramolecular structures formed by d[CCG]n repeat sequences arise (i) from conformational fluctuation of C-C mismatched base pairs within the hairpin stem (Figure 1.12A) (Gao et al., 1995; Zheng, 1996; Mariappan et al., 1998; Romero et al., 1998), (ii) from the different alignments of individual d[CCG]n hairpins (Figure 1.12B) (Chen, 1995; Mariappan et al., 1996b, 1998; Zheng et al., 1996; Yu et al., 1997; Darlow and Leach, 1998a,b), and (iii) from the global organization of multiple hairpins (or other structures) within the larger secondary structure (slipped DNA, or S-DNA) (Figure 1.12C), as first described by Sinden and co-workers (Pearson et al., 1998; Peyret at al., 1999). The first source of conformational mobility for d[CCG]n lies in the C-C mismatched base pairs (Figure 1.12A). The anti-parallel C-C mismatch is one of the Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 29 least stable mismatched pairs (Peyret, 1999) and is correspondingly one of the more conformationally flexible. The C-C mismatch has been found to be intrahelical in otherwise Watson-Crick duplexes in solution (Boulard et al., 1997) and in the solid state (Brown et al., 1990). A similar intrahelical location has been shown by Mariappan et al. (1998) for the C-C pair within a d[GCC]n hairpin. In contrast, Gao et al. (1995) have shown that the duplex d[CCGCCG] 2 forms a structure with 5'- cytosine overhangs and a central d[GCC]«d[GCC] sequence in which the cytosines of the C-C mismatched pair adopt an extrahelical location in the minor groove. C oC C oC C oC B G • C C oC C • G G * C C oC C * G G • C 5 ' C C • G C oC G • C C • G C oC G • C C ® G C oC G ® C 5 ' C • G C oC G * C C G C G C C C C G C C C C • G G • C C oC C • G G • C C C G C C C G C C G G • C C C C C C * G G ® C C oC C * G G * C 5 ' Figure 1.12. Schematic representation of the possible conformers of single strand of d[CCG]n, showing molecules containing Watson-Crick pairs (•) and C-C mismatch pairs (o). A. Intrahelical and extrahelical C-C mismatched pairs within the hairpin stem. B. Alignments of d[CCG]n hairpin stems. C. Slipped DNA (S-DNA) form of d[CCG]n containing multiple hairpins. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 30 Two alignments, both containing a G-C pair, a C-G pair and a C-C mismatched pair within a repeat fragment, are possible for the d[CCG]n hairpin stem (Yu et al., 1997; Darlow and Leach, 1998a, 1998b). These alignments contain either a d[GC]«d[GC] or a d[CG]*d[CG] Watson-Crick dinucleotide step (Figure 1.12B). Hairpins formed from small numbers of d[CCG] repeats have stems containing d[GC]*d[GC] steps, even if formation of this alignment requires the simultaneous formation of an overhanging end (Mariappan et al., 1998), but longer hairpins may adopt the alternative alignment (Yu et al., 1997). In addition, longer triplet repeat sequences form structures (S-DNA) that probably contain multiple hairpins (Pearson and Sinden, 1996; Pearson et al., 1998). For d[CCG]n, such structures would have many C-C mismatched pairs, perhaps in a dynamic equilibrium between intrahelical and extrahelical locations (Zheng et al, 1996), and could also contain hairpins in various alignments (Figure 1.12C). 1.8 Mechlorethamine DNA Crosslinking Reaction Mechlorethamine (Figure 1.13 A) is a nitrogen mustard that can react with nucleophillic sites of DNA such as N7 of guanine (Mattes et al., 1986; Kohn et al., 1987) and with N3 of adenine (Pieper and Erickson, 1990; Wang et al., 1994; Wang et al., 1991). Such reaction occurs via an aziridinium ion intermediate (Rutman et al., 1969, Povirk and Shuker, 1994). The antitumor activity of mechlorethamine and other nitrogen mustards may be due to interstrand DNA crosslinks at two guanine Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 31 bases in DNA duplexes containing d[GiXC] *d[G3 YC], where X-Y is a Watson-Crick pair (Rink and Hopkins, 1995; Rink et al., 1993; Hopkins, 1991; Ojwang et al., 1989). This crosslinking reaction results in the crosslinking of the G1-G3 guanine bases in the major groove of the DNA duplex (Figure 1.13B and D). The 1,3 G-G crosslink product has been shown to form preferentially over the apparently geometrically more favorable 1,2 G-G crosslink (Millard etal., 1990). Figure 1.13. Mechlorethamine (a) and representations of DNA interstrand crosslinks induced by mechlorethamine at (b) a 1,3 G-G site and (c) a C-C mismatch pair. X and Y represent Watson-Crick base pairs. Figures (d) and (e) show the probable connectivities of the mechlorethamine crosslink through (d) the guanine N7 atoms at a 1,3 G-G site and (e) the cytosine N3 atoms of a C-C mismatch pair. Sugar (e) Sugar Y X 5' (C) Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 32 In 1999, Romero et al. showed that mechlorethamine can also form an interstrand DNA crosslink at a C-C mismatched pair (Figure 1.13C). It was also shown that the mechlorethamine C-C crosslink formation occurs regardless of the base sequence flanking the C-C mismatch (Romero et al., 1999). Although the detailed structure of the crosslinked duplex has not yet been determined, it is probable that the reaction occurs in the DNA minor groove through cytosine N3 (Figure 1.13E) (Romero et al., 1999). Within a d[GCC]*d[GCC] sequence the mechlorethamine C-C crosslink forms in preference (Romero et a l, 1999) to the better known 1,3 G-G interstrand crosslink (Rink and Hopkins, 1995; Rink et al., 1993; Millard et al., 1990; Ojwang et al., 1989). The discovery of mechlorethamine C-C crosslinking reaction suggests that mechlorethamine may be used to probe structures formed by d[CCG]n containing cytosine-cytosine mismatched pairs. 1.9 Dissertation Overview The goal of this dissertation is to identify the structures of C-C mismatched pairs within DNA containing d[GCC]n *d[GCC]n repeats, using the mechlorethamine C-C crosslinking reaction as a chemical probing reaction. To accomplish this goal the mechlorethamine C-C crosslinking reaction was first characterized in Chapters 2 and 3. The kinetics of the mechlorethamine crosslinking reaction at a single C-C mismatched pair within DNA was determined in Chapter II. In this chapter, the effects of sequences flanking the C-C mismatched pair on the crosslinking reaction Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 33 were also examined. In Chapter III, the mechlorethamine C-C crosslinking reaction with DNA containing multiple C-C mismatched pairs was investigated. In addition, the electrophoretic mobility of the crosslinked duplexes on denaturing polyacrylamide gels was studied in order to identify the source of this mobility. After obtaining the appropriate crosslinking reaction and suitable separation conditions, mechlorethamine was used to determine possible structures formed by a series of DNA duplexes containing C-C mismatched pairs within d[GCC]n«d[GCC]n repeats (Chapter IV). The findings of intrahelical and extrahelical cytosines within DNA duplexes in Chapter IV extend the dissertation to examine designed molecules that can influence the equilibrium between the intrahelical and extrahelical bases. The effects of spermine and cytosine methylation on the equilibrium between the intrahelical and extrahelical bases were examined in Chapter V and 6. In order to probe directly the hairpin structures formed by single-stranded d[CCG]n using mechlorethamine C-C crosslinking reaction, the single-stranded d[GCCGCCGCCGCCGCC] (dfGCCJs) is, therefore, used to test whether or not mechlorethamine can crosslink hairpin structures (Chapter VII). In Chapter VIII, an HPLC method was developed to provide an alternative method for separation and purification of crosslinked DNA. This method would facilitate collecting large amounts of the sample for further structural analysis using M.S. and N.M.R. techniques. Finally, the findings in this disseration research were summarized and Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 34 the biological significance of eE-DNA conformation in the developent of Fragile X syndrome were discussed in the in Chapter IX. 1.10 Dissertation Significance The significance of the research is both biophysical and biological. The C-C mismatched pairs are very flexible and have potential to induce the formation of unusual DNA structures. Due to the complexity of DNA conformations containing C-C mismatches, the development for an efficient method to identify these structures is important. Identifying the structures of C-C mismatched pairs within a single stranded d[CCG]n may help in the analysis of their involvement in DNA biochemical processes such as replication, repair, and recombination. These relationships will contribute to an understanding of molecular mechanisms of expansions and hypermethylation of d[CGG]n »d[CCG]n triplet repeats associated with Fragile X syndrome. Here, the mechlorethamine crosslinking reaction at a C-C mismatched pair is developed as a probing reaction for DNA triplet repeats containing C-C mismatched pairs. The reaction is capable of detecting extrahelical cytosine bases within d[GCC]n»d[GCC]n repeat fragments, providing evidence o f an extended E- motif DNA (eE-DNA) conformation, which may be responsible for the development of Fragile X syndrome. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 35 CHAPTER II DNA Interstrand Crosslink Formation by Mechlorethamine at a Cytosine-Cytosine Mismatched Pair: Kinetics and Sequence Dependence 2.1 Introduction Expansion of the triplet repeat DNA sequence d[CGG]n«d[CCG]n is a characteristic of Fragile X syndrome, a human neurodegenerative disease (Wells, 1996; Gellibolian, 1997; Mitas, 1997; Pearson and Sinden, 1998; Gacy and McMurray, 1998; Sinden, 1999). Stable intrastrand hairpins formed by both d[CGG]n and d[CCG]n, and involving G-G and C-C mismatched pairs, respectively, are believed to be of importance in the development of the disease (Figure 2.1) (for a review see Darlow and Leach; 1998a, 1998b). For d[CCG]n, hairpins adopt two possible alignments containing either d[CCG]n«d[CCG]n or d[GCC]n »d[GCC]n repeats within the stem as described in Chapter 1 (Figure 2.1). In 1999, Romero et al. showed that a C-C mismatched pair could be crosslinked covalently by mechlorethamine, a nitrogen mustard alkylating agent Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 36 (Figure 2.2). They also demonstrated that, within a d[GCC]»d[GCC] sequence, the mechlorethamine C-C crosslink forms in preference to the better known 1,3 G-G interstrand crosslink (Figure 2.2) (Ojwang et ah, 1989; Hopkins, 1991; Rink et al, 1993; Rink and Hopkins, 1995). Hence, the mechlorethamine C-C crosslinking reaction may be of value as a probe for conformers of d[CCG]„ containing C-C mismatched pairs. A C • G C o C G * C C • G C o C G * C C • G Co C G • C 5 ' B Figure 2.1. Schematic representation of two possible alignments of d[CCG]n, showing molecules containing Watson-Crick pairs (•) and C-C mismatch pairs (o). A. A d[CCG]n«d[CCG]n alignment contains a d[GC]*d[GC] Watson-Crick dinucleotide step. B. A d[GCC]n «d[GCC]n alignment contains a d[CG]»d[CG] Watson-Crick dinucleotide step. ch3 C , ^ N X ^ C 1 (a) CH3 5. X I Y c A / N V \ c Y X 5 ' (c) Figure 2.2. Mechlorethamine (a) and representations of DNA interstrand crosslinks induced by mechlorethamine at (b) a 1,3 G-G site and (c) a C-C mismatch pair. X and Y represent Watson-Crick base pairs. CH3 5 ' C I r — G 3 X Y G ,-1 C 5 ’ (b) G • C C o C C • G G • C C o C C • G G • C 5 ' C A Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 37 In defining the appropriate conditions for the probing reactions, the reaction at a single C-C mismatched pair was first examined in more detail, using a series of model duplexes. In this chapter, the kinetics of the reaction were performed. The results indicate that the reactivity of the C-C mismatch towards mechlorethamine increases with increasing G-C content of the base pairs flanking the C-C mismatch. In addition, the data also show that the electrophoretic mobility of the crosslinked duplex is similarly dependent on the G-C content of the flanking sequence, and that these results are related to the local stability of the duplex around the C-C mismatched pair. 2.2 Materials and Methods Chemicals: Mechlorethamine [bis(2-chloroethyl)methylamine, nitrogen mustard] and T4 polynucleotide kinase were purchased from Sigma. [y-3 2 P]ATP was purchased from ICN. All synthetic oligodeoxyribonucleotides were synthesized on an Applied Biosystems Model 394 automated synthesizer, deprotected, and purified with a COP cartridge at the USC Norris Cancer Center at the University of Southern California. All other reagents were at least analytical grade. 3 2 P-5'-end Labeling o f DNA: Approximately 10pg of column purified synthetic DNA was 5'-end labeled with [y-3 2 P]ATP (5pi, 4500 Ci/mmol) by incubation in buffer (30mM Tris (pH 7.8), 10 mM MgCb, 5mM dithiothreitol) and Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 38 30 units of T4 polynucleotide kinase for 1 hour at 37°C. The reaction was stopped by addition of 3M sodium acetate (5.5pL, pH 5.2) and pre-chilled 95% ethanol (150 pL). The unincorporated [y-3 2 P]ATP was removed by precipitation in 95% ethanol at -20°C overnight, lyophilized, and resuspended in a 0.1M NaCl solution. Alkylation o f DNA: An equal amount of the unlabeled complementary strand was added to a 0.1M NaCl solution of the labeled oligodeoxyribonucleotide, heated to 65°-70°C and then slowly cooled to room temperature. Following annealing of the strands, a lpM duplex DNA solution containing 0.1M NaCl and lOmM Tris (pH 7.5) was incubated for various times at 37°C with lOOpM mechlorethamine in a total volume of lOOpL. For each experiment, a fresh solution of lOOmM mechlorethamine was prepared in dimethyl sulfoxide (DMSO), rapidly diluted to lOmM and immediately added to the DNA solution. Following incubation, the reaction was terminated by addition of 3M sodium acetate (5.5pL), tRNA (5mg/mL, 5 pL), and pre-chilled 95% ethanol (150pL), and precipitated in three times the volume of pre chilled 95% ethanol at -20°C overnight, washed, and then lyophilized. The DNA was then dissolved in distilled water (2pL) and tracking dye (8pL, 80% formamide, lmM EDTA, 0.025% bromophenol blue and xylene cyanol). Detection o f Crosslinked DNA: The samples were loaded onto a 20% denaturing polyacrylamide gel (29:1 acrylamide/bisacrylamide, 8M urea, 89mM Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 39 Tris-borate (pH 8.5) and 2mM EDTA (TBE buffer), 0.4mm thick, 38 x 31 cm, 2500 V, 45 W) until the xylene cyanol marker had migrated 15-18 cm. The band due to the mechlorethamine crosslinked DNA was recovered from the gel and sequenced (Romero et al., 1999) to determine the crosslinking site. Quantification and Kinetic Analysis o f DNA Crosslink Formation: After the gel was exposed to X-ray film, the intensity of the crosslink was analyzed and expressed as a percentage of the total DNA in each lane of the gel. Averaged band intensities from three experiments were plotted against incubation time, and the data fitted to the differential equation, -ln[l- (yt/ymax)] = kt*t, where yt is the percentage of crosslink at time t and ym a x is the total crosslink, to obtain the rate constant kt. The rate of total crosslink formation was then fitted to the 1st order rate equation yt= ym a x * (1 - e~k t). Melting Temperature o f DNA Duplexes: All absorption measurements were made with a Shimadzu UV-visible spectrophotometer model UV160U and a 1-cm cuvette. Duplexes at 10 pg/ml or 20 pg/ml were annealed via heating to 90°C for 1 minute in Tris buffer (pH 7.5, 0.1M NaCl, 0.01M Tris) and slow cooling back to ambient temperature. Thermal denaturation profiles were then measured by monitoring absorbance at 260 nm at various temperature intervals. The solutions were allowed to equilibrate at each temperature for 15 minutes before measuring the Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 40 absorbance. The absorbance was corrected for volume expansion and the melting temperature profile was determined by plotting absorbance against temperature. The peak of the first derivative plot of the melting temperature profile was defined as the melting temperature. Molecular Dynamics Simulations: Simulations were performed using the AMBER 4.0 force field (Weiner et al., 1986; Pearlman, et al., 1991) on the 13-mer helices, d[TCACAGCCTGGTT] «d[ AACC AGCCTGTGA] and d[TCACAACTTG- GTT]«d[AACCAACTTGTGA], both of which have a central C-C mismatched pair. Canonical B-DNA helices were first relaxed in a 4000-step conjugate gradient energy minimization, prior to solvation in a box of TIP3P water molecules (Jorgensen et al., 1983). The water box had dimensions 59A x 40A x 40A and a minimum depth of 8A from the solute to the edge of the box. Sodium counterions were then added by evaluating the electrostatic interaction energy of the DNA with a +1 point charge located at the coordinates of the oxygen of each water molecule, and replacing the water molecule at the point of most negative electrostatic potential with a counterion (Esposito et al., 1988). This process was then repeated (with inclusion of the interactions of previously placed sodium ions) until the required number of counterions (sixteen) had been added to achieve two-thirds electrical neutrality. Following brief minimizations of the water and counterions, the entire system was heated for 2ps from 0 to 25K, and then pre-equilibration of the solvent was Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 41 performed for 40ps. A 200ps simulation of the entire system was then performed in the nPT ensemble, using a time step of 0.002ps and a non-bonded cutoff of 8 A, at a temperature of 298K. The temperature was increased linearly from OK to 298K in the first lOps of the simulation. The coordinates of structures generated in the trajectory were saved every 0.4 ps. 2.3 Results 2.3.1 The mechlorethamine C-C crosslink forms more rapidly than the 1,3 G-G crosslink, and reaches a higher final yield To examine the rate of formation of the mechlorethamine DNA interstrand crosslink at a C-C mismatched pair, and to compare this with the rate of formation of the 1,3 G-G interstrand crosslink, the time courses of mechlorethamine crosslinking of DNA duplexes la and lb (Table 2.1) were measured. These duplexes are of identical sequence except for the central base pair, which is a C-C mismatched pair in duplex la and a C-G base pair in duplex lb. Hence, duplex la has both a C-C mismatch crosslinking site and a 1,3 G-G crosslinking site, while duplex lb contains only a 1,3 G-G site. The results of incubations from zero to 120 minutes of duplexes la and lb with mechlorethamine are shown in Figure 2.3A, and a kinetic analysis of these data is shown in Figure 2.3B and Figure 2.3C. This analysis gave a rate constant of 0.05mm1 for the C-C crosslink formation in duplex la, and 0.02mm 1 for Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. the 1,3 G-G crosslink formation in duplex lb (Table 2.1). It is also apparent from Figure 2.3B that the C-C crosslink reaches a final yield which is much greater than that of the 1,3 G-G crosslink. Table 2.1. Kinetic parameters for mechlorethamine crosslinking of duplexes containing a C-C mismatch crosslink site and a 1,3 G-G crosslink site. Duplex Sequencea Crosslink Rate constant, kt (% crosslinks min'1 ) b % Crosslink b ’c Ia 5 ' - CTCTCACAGCCTGGTTCAG GAGAGTGTCCGACCAAGTC-5 ' C-C 0.05 ± 0.01 27.5 lb 5 ' -CTCTCACAGCCTGGTTCAG GAGAGTGTCGGACCAAGTC- 5 ' 1,3 G-G 0.02 ± 0.008 10.0 a The positions of the crosslink is indicated in each duplex, with cross inked bases shown in bold. b Average values obtained from three separate experiments. c The amount of crosslinked DNA expressed as a percentage of the total DNA. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 43 0 5 10 15 20 30 40 60 120 0 5 10 15 20 30 40 60 120 X —» (C-Q - n - <— X (13 G-G) ■M ■ S B 3 2 1 0 0 10 20 30 40 50 60 tfl 20 10 0 0 20 40 60 80 100 120 Time(min) Time(min) Figure 2 .3 . Kinetics of mechlorethamine DNA interstrand crosslink formation for reaction times up to 120 minutes. A. Autoradiogram of a 20% DP AGE gel following incubation of duplexes la and lb (Table 2.1) with 100pM mechlorethamine. For each duplex lane 0 is a control (no mechlorethamine) and all other lanes show the products of incubation for the time (in minutes) indicated above the lane. For duplex la a C-C crosslink band is observed (band X (C-C)), and for duplex lb a 1,3 G-G crosslink band is observed (band X (1,3 G-G)). Bands due to monoadducts and unreacted single strands are identified as M and S, respectively. B. Quantification of the autoradiograms showing the time course of total crosslink formation following incubation with lOOpM mechlorethamine of duplexes la (A, C-C crosslink) and lb (□, 1,3-G-G crosslink). C. Linearization of the time course data for crosslink formation in duplexes la (A, C-C crosslink) and lb (□, 1,3-G-G crosslink), showing a plot of -ln[l-(yt/ym a x)] vs. kt*t, where t = time, ym a x is the steady state percentage crosslinking, yt is the percentage crosslink at time t, and kt is the first order rate constant for crosslink formation. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 44 2.3.2 The mechlorethamine C-C crosslink is more stable than the 1,3 G-G crosslink The products of incubation of mechlorethamine for up to 24 hours with duplexes la and lb (Table 2.1) are shown in Figure 2.4A, and the band intensities from the gel are plotted against time in Figure 2.4B. For the C-C crosslink in duplex la, the final yield of 27.5% of the total DNA attained after 2 hours is still maintained at 24 hours (Figure 2.4B). In contrast, the maximum level of the 1,3 G-G crosslink in duplex lb (about 10% of the total DNA after 2 hours) decreased to only 6 % after 24 hours (Figure 2.4B). 2.3.3 The amount of mechlorethamine C-C interstrand crosslink formed is dependent on the GC:AT content of the base pairs flanking the C-C mismatch It was shown that mechlorethamine could form an interstrand crosslink at any C-C mismatched pair, regardless of the flanking sequence (Romero et al., 1999). However, in performing these experiments, it was noticed that the amount of crosslink product was variable in a series of duplexes Ha - Ilk (Table 2.2), and that the electrophoretic mobility of the band due to the crosslinked DNA also varied with the duplex sequence (Romero et al., 1999). These data are quantified in Table 2.2 (note that duplex na is the same as duplex la). The amount of crosslinked DNA as a percentage of the total DNA (Table 2.2) decreased with a decreased GC:AT ratio Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 45 within the base pairs flanking the C-C mismatch (M2 -m2 , Mi-mi, m-Ni and n2 -N2 ). Hence, for duplexes Ha, lib and lie, an average of 27.2% of the total DNA was crosslinked (Table 2.2), whereas < 20% of the total DNA was crosslinked in duplexes Hi and Ilj (Table 2.2). The duplexes having an intermediate GC:AT ratio in the base pairs flanking the C-C mismatch (duplexes lid, He, Ilf, and Ilg) gave an average of 26.3% crosslinked DNA (Table 2.2). Although the difference in crosslinking efficiency between these duplexes and duplexes Ha, lib and lie is small, a trend of reduced crosslinking efficiency with reduced G-C content is apparent. It is also noted that there is reduced crosslink formation with duplex Ilk, compared to duplexes Ha, lib and lie. All four of these duplexes have identical GC:AT ratios within base pairs M 2 -m2 , Mi-mi, ni-Ni and n2 -N2 , but the G-C pairs are distal to the C-C mismatch in duplex Ilk (Table 2.2). Based on this observation, the proximity of G-C base pairs to the C-C pair determines the level of crosslinking, in addition to the GC:AT ratio of the flanking sequences. It is noted that other structural features present in a given duplex could also influence the crosslinking efficiency. Such effects have been previously observed (Esposito et al., 1988), and may be the explanation for the relatively high level of crosslinking observed with duplex Ilh, compared to duplexes Hi and Ilj. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 46 X—► (C-C) 01 2 34 5 610152024 0 1 2 3 4 5 610152024 •isiiiivff " n -4—x (1,3 G-G) -M -S B 30 k k k k k Time(hr) Figure 2.4. Kinetics of the mechlorethamine DNA interstrand crosslink formation for reaction times up to 24 hours. A. Autoradiogram of a 20% DP AGE gel following incubation of duplexes la and lb (Table 2.1) with lOOpM mechlorethamine. For each duplex lane 0 is a control (no mechlorethamine) and all other lanes show the products of incubation for the time (in hours) indicated above the lane. For duplex la both a C-C crosslink band (band X (C-C)) and a 1,3 G-G crosslink band (not labeled, but appearing after 6 hours incubation of duplex la, with a slightly greater mobility than the C-C crosslink band) are observed, and for duplex lb a 1,3 G-G crosslink band (band X (1,3 G-G)) is observed. Bands due to monoadducts and unreacted single strands are identified as M and S, respectively. B. Quantification of the autoradiograms showing the time course of total crosslink formation following incubation with lOOpM mechlorethamine of duplexes la (A, C-C crosslink) and lb (□, 1,3-G-G crosslink) for incubation times up to 24 hours. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 47 Table 2.2. Mechlorethamine C-C mismatch crosslink formation3 , electrophoretic mobility of the crosslinked duplex, and duplex melting temperatures (Tm ). 5 " - CTCTCACM2 M iCn1 n 2GGTTCAG i GAGAGTGm2 m iCNiN2CCAAGTC - 5 " % Crosslink Mobility (cm) r p b J -m (°C) Ila A G C C T 1 T C C G A 27.5 14.6 54.5 lib A C C G T 1 T G C C A 27.0 14.6 * lie A C C C T 1 T G C G A 27.0 14.6 54.5 lid A T C C T 1 T A C G A 26.7 14.4 * He A C C T T 1 T G C A A 26.5 14.4 * Ilf A G C T T 1 T C C A A 26.0 14.4 53.8 Hg A T C G T 1 T A C C A 25.9 14.4 * Ilh A T C T T 1 T A C A A 25.0 14.2 52.9 Hi A T C A T 1 T A C T A 20.9 14.2 51.2 Hj A A C T T 1 T T C A A 18.2 14.2 ** Ilk G A C T C i C T C A G 25.4 14.4 52.3 The positions of the crosslink is indicated in eac l duplex, with crosslin <ed base shown in bold. b Based on an average of three experiments. * Tm was not measured. ** Tm could not be accurately measured due to the flatness of the absorbance curve. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 48 2.3.4 The electrophoretic mobility of the mechlorethamine C-C crosslinked species is dependent on the GC:AT content of the base pairs flanking the C-C mismatch The mobilities of the crosslinked duplexes Ha - Ilk on a denaturing polyacryl amide gel were also quantified and these data are given in Table 2.2. It is apparent that the GC:AT ratio of the flanking base pairs (M2 -ni2 , Mi-mi, n t-Ni and n2 -N2 ) influences this mobility, and that greater G-C content leads to a greater electrophoretic mobility. This mobility is also a function of the sequence of the flanking base pairs, as shown by comparison of duplexes Ha and Ilk (Table 2.2). 2.3.5 Sequential replacement of A-T pairs by G-C pairs has a predictable effect on the amount of C-C crosslinked DNA and on the electrophoretic mobility of the crosslink To test further the effect of the GC:AT ratio and the proximity of G-C pairs to the C-C mismatch on the formation and electrophoretic mobility of the crosslink, the duplex sequences shown in Table 2.3 are used. The autoradiogram of the denaturing gel of the products of incubation of these duplexes with mechlorethamine are shown in Figure 2.5. The band mobilities and the amount of crosslink formed are shown in Table 2.3. Increasing the GC:AT ratio of the four base pairs to each side of the C-C mismatch (duplexes Mb, IIIc, Hid and Me, compared to duplex Ma, Table Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 2.3) increases both the amount of crosslink and the electrophoretic mobility of the crosslinked species. For the four duplexes (Illb, IIIc, Hid and Ille), which have identical GC:AT content in the flanking base pairs, as the G-C pair becomes more proximal to the C-C mismatch the amount of crosslink and the electrophoretic mobility of the crosslinked duplex increases. Table 2.3. Mechlorethamine C-C mismatch crosslink formation3 and electrophoretic mobility of the crosslinked duplex. 5 ' -CTCCCM4 M 3 M 2 M1 C n 1 n 2 n 3 n 4CCCAG % Mobility 1 GAGGGrrumsmsmiC N iN 2 N 3 N4 GGGTC~ 5 ' Crosslink (cm) Ilia A A T T C A A T T 1 T T A A C T T A A 8.7 11.5 Illb GA T T C A A T C I C T A A C T T A G 9.2 1 2 . 8 IIIc A G T T C A A C T 1 T C A A C T T G A 12.5 13.2 Hid A A C T C A G T T 1 T T G A C T C A A 12.9 13.4 Ille A A T C C G A T T 1 T T A G C C T A A 16.9 13.6 a The positions of the crosslink is indicated in each duplex, with crosslinked bases shown in bold. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 50 1 2 3 4 5 6 7 8 9 10 Ilia Illb HIc Hid nie < M <— S Figure 2.5. Autoradiogram of a 20% DP AGE gel following incubation for 6 hours with 100pM mechlorethamine of duplexes Ilia to Ille (Table 2.3). In lanes 1-10, the odd numbered lanes are controls (no mechlorethamine) and the even numbered lanes show the products resulting from incubation of the indicated duplex with mechlorethamine. Bands are identified as X (C-C crosslink), M (monoadduct), and S (unreacted single strands). Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 51 2.3.6 Molecular dynamics simulations suggest the C-C mismatched pair is stacked more favorably within a d[GCC]»d[GCC] sequence than in a d[ACT]» d[ACT] sequence To understand the basis for the sequence-dependent properties of the mechlorethamine C-C crosslinked duplex, molecular dynamics simulations were performed on solvated duplexes comprising the central 13 base pairs of duplexes Ha and Ilj, d[TCACAGCCTGGTT]»d[AACCAGCCTGTGA] and d[TCACAACTT- GGTT]»d[AACCAACTTGTGA], referred to as duplexes Ila' and Ilj', respectively. The fluctuation over each simulation trajectory of Watson-Crick hydrogen bond distances in the base pairs neighboring the C-C mismatch was used as a measure of stability, and these data are shown in Figure 2.6B and Figure 2.6D for duplexes Ila' and Ilj', respectively. For duplex Ila', the G-C base pairs flanking the C-C mismatch remain hydrogen bonded and the N3-N3 distance in the C-C mismatched pair is essentially unchanged over the simulation (Figure 2.6 A), suggesting that the C-C mismatched pair in duplex Ila' is stacked within the duplex. In contrast, for duplex Ilj', the hydrogen bonding of the A-T base pairs flanking the C-C mismatch is disrupted, and the C-C mismatch itself is unpaired (Figure 2.6C), causing a local distortion at the center of duplex Ilj'. The additional distortion in duplex Ilj' is apparent from a comparison of structures of duplex Ila' (Figure 2.7A) with duplex Ilj' (Figure 2.7B) after 60 ps of the simulation (Figure 2.7). In duplex Ila' the C-C mismatch remains paired and stacked between two Watson-Crick G-C pairs (Figure Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 52 2.6B, Figure 2.7A), whereas in duplex Ilj' the cytosines of the C-C mismatched pair are beginning to separate, and one of the flanking T-A pairs is no longer hydrogen bonded (Figure 2.6D, Figure 2.7B). a< 12 10 8 6 4 2 0 0) u c 8 tn 50 100 150 200 Time (ps) B .< 12 * 1 0 0) Q o 2 c 6 8 4 « 2 Q 0 D . A im U 50 100 150 200 Time (ps) • < 12 i M m ^ 10 - < D a . c 6 - 8 4 - » 2 - i i ...... i.......... Q 0 - i i i 0 50 100 150 200 Time (ps) 50 100 150 200 Time (ps) Figure 2.6. Data from molecular dynamics simulations of duplexes Ila' and Ilj' (the central 13 base pairs of duplexes Ila and Ilj, Table 2.2), showing the motions of the C-C mismatch pair and the neighboring Watson-Crick base pairs over the simulation. The figure shows the N3 to N3 distance of the C-C mismatch pair for duplex Ila' (A) and duplex Ilj' (C) and the averaged distance of the Watson-Crick hydrogen bonds of the two base pairs neighboring the C-C mismatch pair in duplex Ila' (B) and duplex Ilj' (D). Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 53 Figure 2.7. Structures taken after 60ps of the molecular dynamics simulations of duplex Ila' (A) and duplex Ilj' (B). The central three base pairs, including the C-C mismatch pair, are labeled for each duplex. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 54 2.4 Discussion DNA interstrand crosslink formation by mechlorethamine at a 1,3 G-G crosslinking site is thought to proceed as two pseudo-first order reactions (Rutman et al., 1969). The rate-determining step of the reaction is formation of a cyclic aziridinium ion through loss of chloride from the free mechlorethamine. Rutman et al. (1969) report a rate constant for this step of 0.04 min'1 , while Price (1958) reports a value of 0.02 min' 1 to 0.05 m in1 . The rate constant determined here for the overall rate of C-C crosslink formation, 0.05 min'1 , is similar to these values. Hence, formation of the C-C crosslink may also be rate-limited by formation of the aziridinium ion intermediate. Interstrand crosslinks are formed from the monoalkylated species (the monoadduct) through reaction of the 'second arm' of the mechlorethamine. Alternatively, terminal monoadducts can form due to competing reactions (particularly hydrolysis) of the second arm (Salvati et al., 1992). The rate constant for the second arm reaction for mechlorethamine crosslinking of calf thymus DNA has been reported as 0.08 min' 1 (Rutman et al., 1969). The rapid rate of this reaction, relative to the initial aziridinium ion formation, has been confirmed in a Hindlll pBR322 restriction fragment (Hartley et al., 1991) and in shorter duplexes containing 1,3 G-G crosslink sites (Povirk and Shuker, 1994). Given these numbers, the slower overall rate of formation of the 1,3 G-G crosslink (0.02 min'1 ), compared to the C-C Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 55 crosslink (0.05 min'1 ), may be due to more extensive hydrolysis of the second arm of the guanine monoadduct, and the formation of more terminal monoadducts. It has been previously noted an apparent lack of cytosine monoadducts formed with duplexes containing C-C mismatched pairs (Romero et al., 1999). The amount of the mechlorethamine C-C crosslink formed is reduced by a decreased GC:AT ratio in the bases flanking the C-C mismatched pair, and also, for the same GC:AT ratio, by having G-C pairs distal to the C-C mismatch. Changes in base content and sequence up to four bases from the C-C mismatch have an effect. This may be a consequence of decreased duplex stability (as indicated by the duplex melting temperature, Tm ), or a local opening of the helix near the C-C mismatch, caused by increased numbers of A-T pairs in the flanking region (as suggested by computer simulation). The series of duplexes show the expected relationship of GC:AT ratio to Tm , but the duplex melting temperatures do not vary by more than 3.5°C (Table 2.2). This perhaps suggests that the amount of crosslink formation may be more dependent on local instability and conformational fluctuation around the C- C mismatch, rather than on the overall position of the duplex to single-strand equilibrium. However, it is noted that other factors may play a role in modulating the crosslinking reaction. In particular, previous work addressing the sequence specificity of psoralen crosslink formation suggested that subtle changes in the helical structure, caused by changes in the base sequence flanking the crosslink site, Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 56 could influence the crosslinking efficiency (Esposito et al., 1988). Residual alkylation at neighboring sites may also be of importance. The electrophoretic mobility of the C-C crosslinked duplexes also shows an apparent dependence on the GC:AT content of the sequences flanking the C-C mismatch, and on the proximity of G-C pairs to the C-C mismatch. Hence, local stability at and around the C-C crosslink site leads to a greater mobility of the crosslinked species in the denaturing gel. A somewhat similar phenomenon has been observed for psoralen-crosslinked DNA duplexes (Kumaresan et al., 1992). The rapid kinetics of formation of the C-C crosslink and, in particular, its subsequent resistance to degradation provides confidence that the reaction will be useful to probe the C-C mismatched pairs in DNA conformers of d[CCG]n repeats. The results also suggest that different C-C mismatched pairs in such structures may exhibit different crosslinking efficiency, depending on their location (for example, their position relative to a hairpin loop), and this may be of value in further defining a complex DNA conformation. The subtle dependence of electrophoretic mobility on local structural stability and crosslink location should also be of value in the separation of the crosslinked species. The electrophoretic mobility of crosslinked duplex will be extensively investigated in the next chapter. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 57 CHAPTER III DNA Interstrand Crosslink Formation by Mechlorethamine at a Cytosine-Cytosine Mismatched Pair: Electrophoretic Mobility of the Crosslinked Duplexes 3.1 Introduction Fragile X syndrome is characterized by large expansions of the triplet repeat DNA sequence d[CGG]n*d[CCG]n (Fu et al., 1991; Tapscott et al., 1998) within 5'- untranslated region of the Fragile X Mental Retardation 1 (FMR1) gene. The development of the disease is believed to be due to intrastrand conformations formed by both d[CGG]n and d[CCG]„ (Darlow and Leach, 1998a, 1998b). d[CCG]„ forms stable intrastrand hairpin DNA conformations containing C-C mismatched pairs (Darlow and Leach, 1998a). A potential approach to study the molecular details of d[CCG]n repeats is to chemically crosslink the cytosine bases within the repeats by mechlorethamine (Figure 3.1). To accomplish this approach, the mechlorethamine C- C crosslinking reaction was investigated by examining the kinetics and sequence dependence of the reaction (Chapter II). In performing this work, anomalies were noticed in the electrophoretic mobilities of different crosslinked duplexes, and these are investigated further in this chapter. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 58 A c h 3 c 1 a / n v \ c 1 Sugar Sugar Figure 3.1. A. Mechlorethamine and B. a representation of the DNA interstrand crosslink induced by mechlorethamine at a C-C mismatched pair, showing the probable connectivities of the mechlorethamine crosslink through the cytosine N3 atoms of the mismatched pair. This chapter demonstrates that mechlorethamine can crosslink a C-C mismatched pair at various positions on duplex DNA and even multiple, isolated and contiguous, mismatched cytosines within the Fragile X sequence. However, the crosslinked duplexes show unexpected variable mobility on denaturing polyacrylamide gels. The source of this mobility is then explored by examining the mobility of the C-C crosslink in various positions within duplex DNA that does not contain the Fragile X sequence. The results show that the mobility of these crosslinked duplexes is a function of the location of the crosslinked C-C pair. It is also shown that the differential mobilities of identical duplexes carrying either top or bottom strand labels is a function of the label itself. These results are explained by considering a combination of base content, local duplex stability, and the influence of the label. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 59 3.2 Materials and Methods Chemicals'. Mechlorethamine [bis(2-chloroethyl)methylamine)] and T4 poly nucleotide kinase were purchased from Sigma. 5 '-fluorescein phosphoramidite ([(3',6'-dipivaloylfluoresceinyl)-6-carboxamidohexyl]-l-0-(2-cyanoethyl)-(N,N-di- isopropyl)phosphoramidite) was purchased from Glen Research. [y-3 2 P] ATP was purchased from ICN. All synthetic oligodeoxyribonucleotides were synthesized on an Applied Biosystems Model 394 automated synthesizer, deprotected, and purified with a COP cartridge and, when required, labeled with the 5'- fluorescein phosphoramidite. The oligodeoxyribonucleotides were synthesized at the USC Norris Cancer Center at the University of Southern California. All other reagents were at least analytical grade. 3 2 P- 5 '-end labeling o f DNA: Approximately lOpg of column purified synthetic DNA was 5'-end labeled with [y-3 2 P]ATP (5pi, 4500 Ci/mmol) by incubation in buffer (30mM Tris (pH 7.8), 10 mM MgC12, 5mM dithiothreitol) and 30 units of T4 polynucleotide kinase for 1 hour at 37°C. The reaction was stopped by addition of 3M sodium acetate (5.5pl, pH 5.2) and pre-chilled 95% ethanol (150 pi). The unincorporated y3 2 P-ATP was removed by precipitation in 95% ethanol at -20°C overnight, lyophilized, and resuspended in a 0.1 M NaCl solution. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 60 Alkylation o f DNA: An equal amount of the unlabeled complementary strand was added to a 0.1 M NaCl solution of the labeled oligodeoxyribonucleotide, heated to 65°-70°C and then slowly cooled to room temperature. Following annealing of the strands, a lpM duplex DNA solution containing 0.1M NaCl and lOmM Tris (pH 7.5) was incubated for various times at 37°C with lOOpM mechlorethamine in a total volume of lOOpL. For each experiment, a fresh solution of lOOmM mechlorethamine was prepared in dimethyl sulfoxide (DMSO), rapidly diluted to lOmM and immediately added to the DNA solution. Following incubation, the reaction was terminated by addition of 3M sodium acetate (5.5pL), tRNA (5mg/mL, 5 pL), and pre-chilled 95% ethanol (150pL), and precipitated in three times the volume of pre chilled 95% ethanol at -20°C overnight, washed, and then lyophilized. The DNA was then dissolved in distilled water (2pL) and tracking dye (8 pL, 80% formamide, ImM EDTA, 0.025% bromophenol blue and xylene cyanol). Detection o f Crosslinked DNA: The samples were loaded onto a 20% denaturing polyacrylamide gel (29:1 acrylamide/bisacrylamide, 8 M urea, 89mM Tris-borate (pH 8.5) and 2mM EDTA (THE buffer), 0.4mm thick, 38 x 31 cm, 2500 V, 45 W) until the xylene cyanol marker had migrated 15-18 cm. Bands due to 5'- fluorescein phosphoramidite labeled DNA molecules were detected using UV light and photographed with a Polaroid camera. Bands due to 5 '-3 2 P-phosphate labeled DNA molecules were detected by autoradiography. The bands due to the Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 61 m echloreth am i n e-crossl inked DNA was recovered from the gel and sequenced (Romero et al., 1999) to determine the crosslinking site. 3.3 Results 3.3.1 The mechlorethamine C-C crosslink forms in DNA duplexes with single or multiple C-C mismatched pairs DNA duplexes Ix, ly, Iz, IIx, Ily and III (Table 3.1) were labeled on the ‘top’ strand with 5 '-3 2 P-phosphate and incubated with mechlorethamine. The products of the reaction were separated by electrophoresis on a denaturing polyacrylamide gel (Figure 3.2A). In Figure 3.2A, bands with slower mobility (labeled X) are species that have a mechlorethamine crosslink at a C-C mismatched pair (Romero et al., 1999), except for the band in lane 4 (duplex 0), which is due to a 1,3 G-G crosslinked duplex (Hopkins et al, 1991). Each C-C mismatch crosslink band was sequenced to determine through which ‘top’ strand base the crosslink formed (Figure 3.2C). For duplexes Ix, Iy and Iz (lanes 12, 8 and 14, respectively, Figure 3.2A) sequencing of the crosslink band gave the expected results (lanes labeled Ix, Iy and Iz, Figure 2C). Hence, the crosslink forms through base C7 , C1 0 and C1 3 in duplexes Ix, Iy and Iz, respectively. For duplex IIx two distinct bands (labeled a and b, lane 6, Figure 3.2A) occur. Sequencing confirmed that these bands represent two different crosslinked duplexes in which crosslinks formed through C1 3 and C7 , respectively Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 62 (lanes Ilx/a and Ilx/b, Figure 2C). A similar result was obtained for duplex Ily (lane 10, Figure 3.2A), with crosslinks forming through Cio and C7, respectively (lanes Ily/a and Ily/b, Figure 3.2C). For duplex III three crosslinked products (bands labeled a, b and c, lane 2, Figure 3.2A) form upon incubation with mechlorethamine. Again, sequencing showed these to be due to duplexes with crosslinks formed through Cio, C 13 and C7, respectively (lanes Ill/a, Ill/b and III/c, Figure 3.2C). The relatively weak band consistent with crosslink formation at C1 3 in lane III/c (Figure 3.2C) occurs because of contamination from band Ill/b. It is also noted the presence of other bands in lanes 2 and 10 of Figure 3.2A (labeled The origin of these bands are identified and discussed in Chapter IV (Rojsitthisak et al., 2001). Table 3.1. Electrophoretic mobility of duplexes containing single and multiple C-C mismatch pairs and single mechlorethamine C-C crosslinks a Duplex C-C Crosslink*1 Lane/ Band* Mobility (cm)* GC:AT:CC ‘left’ GC:AT:CC ‘right’ Ix 5 ' -CTCTCGCCGCCGCCGTATC GAGAGCCGCGGCGGCATAG- 5 ' C7 -C32 12 14.4 4: 2:0 9:3:0 iy 5 ' -CTCTCGCCGCCGCCGTATC GAGAGCGGCCGCGGCATAG- 5 ' C i o -C j s 8 14.6 7:2:0 6:3:0 Iz 5 ' -CTCTCGCCGCCGCCGTATC GAGAGCGGCGGCCGCATAG- 5 ' 14 14.2 10 : 2 : 0 3:3:0 IIx 5 ' -CTCTCGCCGCCGCCGTATC GAGAGCCGCGGCCGCATAG- 5 ' C7 -C32 Cl3-C2 e fib fia 13.5 13.0 4: 2:0 9:2:1 8:3:1 3:3:0 ny 5 ' -CTCTCGCCGCCGCCGTATC GAGAGCCGCCGCGGCATAG-5' C7 -C32 C n rQ is 10b 10a 14,4 13.0 4: 2:0 6 : 2 : 1 8:3:1 6:3:0 in 5 ' -CTCTCGCCGCCGCCGTATC GAGAGCCGCCGCCGCATAG- 5 ' c, -c 32 C io -C 2 9 CirC u 2c 2a 2b 11.6 11.4 11.5 4: 2:0 6 : 2 : 1 8 : 2 : 2 7:3:2 5:3:1 3:3:0 0 5 ' -CTCTCGCCGCCGCCGTATC GAGAGCGGCGGCGGCATAG-5 ' none 8d 14.8d - - a Data from Figure 3.2A. b Position of the mechlorethamine C-C crosslink. For duplexes containing multiple C-C mismatch pairs, multiple crosslinked species are observed. 0 G-C, A-T and C-C content of the base pairs to the ‘left’ and to the ‘right’ of the crosslinked C-C mismatch pair. d 1,3 G-G crosslink band. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 63 Figure 3 .2 . A. Autoradiogram of a 20% DPAGE gel showing the products of incubation of duplexes of sequence shown in Table 3.1 with lOOpM mechlorethamine (even numbered lanes) or no mechlorethamine (odd numbered lanes). Each duplex was 3 2 P-radiolabeled at the 5' end of the ‘top’ strand, which is of identical sequence in each duplex. The duplex is identified below the lanes, and the identities of the N7 -N3 2 , N 1 0 -N2 9 and N 1 3 -N2 6 base pairs (C-C or C-G) in each duplex are also shown. Bands are identified as X (C-C crosslink), M (monoadduct), and S (unreacted single strands). The gel was ran until the xylene cyanol marker had migrated 15 cm. B. Autoradiogram of a 20% DPAGE gel showing the products of incubation of duplexes Iy and Ily (Table 3.1) with lOOp'M mechlorethamine. Each duplex carries a 5'-end3 2 P label on either the ‘top’ strand (duplex Iy, lane 8T; duplex Ily, lane 10T) or ‘bottom’ strand (duplex Iy, lane 8B; duplex Ily, lane 10B). C. Autoradiogram of a 20% DPAGE sequencing gel showing the products of Maxam- Gilbert sequencing of the crosslink bands from Figure 3.2A. The lane labeled G shows the results of a Maxam-Gilbert guanine sequencing reaction, and identifies the positions of fragments cleaved at four guanine bases in the ‘top’ strand, which is identical in each duplex. The remaining lanes refer to bands from Figure 3.2A, which are identified by the duplex name and (where necessary) the band letter in Figure 3.2A. In this way, bands corresponding to cleavage of duplexes crosslinked at C1 3 , Cio or C7 can be identified. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 64 A x> M w * S -+ n 7 -n 3 2 ^ 10-^29 n 1 3 -n 2 6 duplex 1 2 3 4 5 6 7 8 9 1011 121314 f - U <—c a b h » > C-C C-G C-C C-G C-C C-C C-G C-C C-G C-G C-C C-C C-G C-G C-C C-G C-C C-G C-G C-G C-C III 0 IIx iy ny Ix Iz B 8 10 T B T B c I C-G C-C C-C C-C C-G C-G iy ny C G Ix Iy Iz in III III a b c IIx a IIx Ily Ily b a b G,s ♦ m * * * ' m c a Gjj •m m m C1 0 g 9 i # * * — C7 G ‘ • Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 65 3.3.2 The mechlorethamine C-C crosslinked DNA duplexes have variable mobility on a denaturing polyacrylamide gel The most striking feature of Figure 3.2A is the variability in the electrophoretic mobility of the crosslinked DNA duplexes. The mobility data are quantified in Table 3.1. For crosslinked duplexes Ix, Iy and Iz, the mobility increases with increased G-C content (increased stability) of the four base pairs on each side of the C-C mismatched pair, as previously described in Chapter II (Romero et al., 2001). Hence, crosslinked duplex Iy has the greatest mobility, followed by Ix and Iz, respectively (Table 3.1). For crosslinked duplex IIx, the relative mobilities of the species giving bands a and b (lane 6 , Figure 3.2A) also follow this relationship. Hence, the duplex crosslinked through C7 (lane Ilx/b, Figure 3.2C) has a greater mobility than that crosslinked through C 1 3 (band Ilx/a, Figure 3.2C), and the G-C content of the bases flanking the C7-C32 mismatched pair is greater than the G-C content of those flanking the C 1 3 -C2 6 mismatched pair (Table 3.1). For crosslinked duplex III, the band of greatest mobility (band a, lane 2, Figure 3.2A) corresponds to the duplex crosslinked through C7 (lane III/c. Figure 3.2C) and the band of intermediate mobility (band b, lane 2, Figure 3.2A) corresponds to the duplex crosslinked through C 1 3 (lane Ill/b. Figure 3.2C). This is consistent with the greater G-C content flanking the C7-C32 mismatched pair (Table 3.1), compared to the bases flanking the C1 3 -C2 6 mismatched pair. The slowest mobility band for duplex III (band a, lane 2, Figure 3.1 A) corresponds to the duplex crosslinked through Cio (lane Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 66 III/a, Figure 3.2C), consistent with the high C-C mismatch content (decreased stability) in the base pairs flanking the crosslinked C-C pair (Table 3.1). However, for crosslinked duplex Ily, the stability of the flanking sequence is not the key determinant of the band mobility. Hence, the band of greater mobility (band b, lane 10, Figure 3.2A) corresponds to the duplex crosslinked through C7 (lane Ily/b, Figure 3.2C) and the slower mobility band (band a, lane 10, Figure 3.2A) corresponds to the duplex crosslinked through Cio (lane Ily/a, Figure 3.2C). This occurs despite the lower G-C content of the bases flanking the C7 -C3 2 pair, compared to those flanking the C1 0 -C2 9 pair (Table 3.1). This result is returned below. 3.3.3 DNA duplexes having an identical mechlorethamine C-C crosslink can have variable DPAGE mobility as a function of the position of the 5'-3 2 p- phosphate label A common method used to identify DNA interstrand crosslinks involves the performance of two separate reactions in which either the ‘top’ strand or the ‘bottom’ strand of the DNA duplex is radiolabeled with 5'-3 2 P-phosphate. The expectation is that the two resulting species will have similar DPAGE mobility (Hartley et al., 1991). When performing this assay with duplex Iy, the expected result was obtained (lanes 8T and 8B, Figure 3.2B), but the same experiment using duplex Ily gave some unexpected results (lanes 10T and 10B, Figure 3.2B). Hence, band a (lane 10T, Figure 3.2B), which results from crosslink formation through Cio (lane Ily/a, Figure Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 67 3.2C) in a duplex carrying a 5'-3 2 P-phosphate label on the ‘top’ strand) and band c (lane 10B, Figure 3.2B), which resulting from an identical crosslink (at C 1 0 -C2 9 ), but T 9 in a duplex carrying a 5'- P-phosphate label on the ‘bottom’ strand, have different mobility. Similarly, when duplex Ily is crosslinked through C7, the DPAGE mobility is dependent on the position of the 5'-3 2 P-phosphate label (contrast band b, lane 10T and band d, lane 10B, Figure 3.2B). 3.3.4 The DPAGE mobility of the mechlorethamine C-C crosslinked duplex depends on the position of the crosslink in the duplex, and on the location of the 5'-3 2 p-phosphate label To determine the basis for the anomalous mobility observed above, several ‘model’ duplexes (Table 3.2) were designed to test the effect on the DPAGE 32 mobility of the position of the crosslink and the position of the 5'- P-phosphate label. Duplexes fVa, IVb and IVc (Table 3.2) have an identical ‘bottom’ strand, but vary in their ‘top’ strands at positions 10, 7 and 4, which are a cytosine or a guanine. Hence, duplex IVa has a centrally located C1 0 -C2 9 mismatched pair, while in duplexes IVb and IVc the C-C pair is shifted towards the duplex end, at C7 -C3 2 and C4 -C3 5 , respectively. The same base pairs neighbor the C-C mismatched pair in each duplex and the duplexes also have identical molecular weights. The electrophoretic mobilities of the mechlorethamine C-C crosslinked duplexes IVa, IVb and IVc carrying a 5 '-3 2 P-phosphate label on either the ‘top’ or ‘bottom’ strand are shown in Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 68 Figure 3.3 A. Two main results are apparent. First, the electrophoretic mobility of the crosslinked duplexes increases from duplex IVa to IVb to IVc. Second, for duplexes IVb and IVc, the location of the 5 '-3 2 P-phosphate label (on the ‘top’ or ‘bottom’ 'i'y strand) also has an influence on the mobility. In each, the species carrying a 5'- P- phosphate label proximal to the crosslink (labeled on the ‘top’ strand) has a slower mobility than the identical crosslinked duplex carrying a 5'- P-phosphate label distal to the crosslink (labeled on the ‘bottom’ strand) (compare lanes 6 and 8 (duplex IVb) or lanes 10 and 12 (duplex IVc) in Figure 3.3A). To confirm these results, further experiments were performed on duplexes Va and Vb (Table 3.2). These duplexes have C-C mismatched pairs at C 1 0 -C2 9 and C7 -C3 2 , respectively, and they also have identical sequences flanking the C-C mismatched pair for six base pairs from the mismatch in each direction, thus eliminating any possible sequence effect on the electrophoretic mobility (Romero et al., 2001). As shown in Figure 3.3B, crosslinked duplex Vb still has greater electrophoretic mobility than crosslinked duplex Va, suggesting that the principal origin of the mobility effect is due to the crosslink position in the duplex. A dependence of mobility on the position of the 5'-3 2 P-phosphate label is also seen for crosslinked duplex Vb, with the species carrying a ‘bottom’ strand label having a faster relative mobility, as for duplex IVb. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 69 Table 3.2. DNA duplexes containing single C-C mismatch pair. Duplex C-C Crosslink GC:AT 'left' GC:AT 'right' IVa 5 ' - CCGGAGGAGCATTCATCTG GGCCTCCTCCTAAGTAGAC-5 " c1 0 -c2 9 7 : 2 3 : 6 IVb 5 ' - CCGGAGCAGGATTCATCTG GGCCTCCTCCTAAGTAGAC-5 ' C7 -C3 2 5 : 1 5 : 7 IVc 5 " - CCGCAGGAGGATTCATCTG GGCCTCCTCCTAAGTAGAC- 5 ' c4 -c3 5 3 : 0 7 : 8 Va 5 ' -CCTATACTCCGAGTATACC GGATATGAGCCTCATATGG-5 " c1 0 -c2 9 4 : 5 4 : 5 Vb 5 ' -ATACTCCGAGTATACCCCT TATGAGCCTCATATGGGGA-5 " c7 -c3 2 2 : 4 6 :6 VI 5 ' - ATTCATCTGCCCGGAGGAG TAAGTAGACCGGCCTCCTC- 5 ' Cio -C2 9 3 : 6 7 : 2 VII 5 " - CCTCGGCCGGATTCATCTG GGAGCCCGCCTAAGTAGAC- 5 ' C7 -C3 2 5 : 1 6 :6 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 70 B duplex 1 2 3 4 5 6 7 8 Va Vb T B T B Figure 3.3. Autoradiograms of 20% DPAGE gels following incubation with lOOpM mechlorethamine of 5'-end 3 2 P-labeled duplexes IVa, IVb, IVc, Va and Vb (Table 3.2). Bands are identified as X (crosslink), M (monoadduct), and S (unreacted single strands). A. Results of mechlorethamine incubation with duplex IVa 3 2 P-labeled on the top strand (lane 2), duplex IVa 3 2 P-labeled on the bottom strand (lane 4), duplex IVb 3 2 P-labeled on the top strand (lane 6), duplex IVb 3 2 P-labeled on the bottom strand (lane 8), duplex IVc 3 2 P-labeled on the top strand (lane 10) and duplex IVc 3 2 P-labeled on the bottom strand (lane 12). Odd numbered lanes from 1 to 11 are controls (no mechlorethamine). B. Results of mechlorethamine incubation with duplex duplex Va 3 2 P-labeled on the top strand (lane 2), duplex Va 3 2 P-labeled on the bottom strand (lane 4), duplex Vb 3 2 P-labeled on the top strand (lane 6), and duplex Vb 3 2 P-labeled on the bottom strand (lane 8). Odd numbered lanes are controls (no mechlorethamine). Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 71 3.3.5 The influence of crosslink position on mobility is unchanged in duplexes carrying a 5'-fluorescein phosphoramidite label, but the influence of the label position on mobility does change Because of the unusual electrophoretic mobility effects of the 5'-3 2 P- phosphate labeled crosslinked duplexes, the experiments were repeated using duplexes IVa, IVb and IVc and 5'-fluorescein phosphoramidite labels. The relative electrophoretic mobilities of the duplexes are unchanged with the new label (that is, the mobility increases from IVa to IVb to IVc, Figure 3.4). However, in other respect the results obtained for the 5'-fluorescein phosphoramidite labeled duplexes are the ‘mirror image’ of those obtained with the 5'-3 2 P-phosphate labeled duplexes (contrast Figure 3.3A with Figure 3.4). Hence, the mobility of crosslinked duplexes IVb (lanes 6 and 8, Figure 3.4) and IVc (lanes 10 and 12, Figure 3.4) is increased when the duplex is labeled on the ‘top’ strand, in contrast to the observations made with the 5'-3 2 P-labeled duplexes (Figure 3.3A). Further, there is also a label- dependent mobility for duplex IVa (lanes 2 and 4, Figure 3.4), despite the centrally located C-C pair and the pseudo-twofold symmetry of the crosslinked species. If this effect is a function of the duplex sequence (GC:AT content) proximal to the label, then a reversal of the sequence of the duplex should result in a reversal of the relative mobilities of the species labeled on the ‘top’ and ‘bottom’ strand. Duplex VI (Table 3.2) has the reverse sequence to duplex IVa (in the sense that the 5' ends of duplex IVa form the 3' ends of duplex VI, and vice versa). Consistent with the effects of the Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 72 5'-fluorescein phosphoramidite label on duplex IVa (lanes 2 and 4, Figure 3.4), the crosslinked duplex VI carrying a label on the ‘bottom’ strand has a greater mobility compared to the same duplex with a ‘top’ strand label (lanes 2 and 4, Figure 3.5A), the reverse of duplex IVa. To show that the effects described above are not due to double-labeling, the mobility of double-labeled duplex VII (Figure 3.5B) was examined. It is apparent that the double-labeled duplex (lane 3, Figure 3.5B) has a significantly retarded mobility, compared to either of the single-labeled duplexes (lanes 2 and 4, Figure 3.5B). 32 3.3.6 Duplexes carrying both 5 '-fluorescein phosphoramidite and 5'- P- phosphate labels show only small differences in duplex mobility as a function of label position The DPAGE mobility of duplexes IVa, IVb and IVc simultaneously labeled with both 5'-fluorescein phosphoramidite and 5'-3 2 P-phosphate is shown in Figure 3.6. In Figure 3.6A the bands are visualized using the 3 2 P radioactivity, and in Figure 3.6B the same gel is visualized using the fluorescence of the fluorescein label. These duplexes show the same increase in electrophoretic mobility as the crosslink is positioned closer to the duplex end (that is, from IVa to IVb to IVc). However, the differential mobility within in each duplex observed for both sets of single-label duplexes (Figures 3.3A and 3.4) is essentially absent. Hence, for example, duplex IVc, having the 5'-3 2 P-phosphate label proximal and the 5'-fluorescein Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 73 phosphoramidite label distal to the crosslink, or the reverse, does not greatly alter the mobility (contrast lanes 10 and 12 in either Figure 3.6A or 3.6B). The same result is also obtained for the other duplexes. It is noted that the method of visualization itself can slightly influence the exact positions of the bands, which are slightly different in Figures 3.6A and 3.6B, despite the figures showing the identical gel. I 1 2 3 4 5 6 7 8 9 10 11 12 I * - ♦ s s duplex Figure 3.4. Autoradiogram of a 20% DPAGE gel following incubation with lOOpM mechlorethamine of 5'-fluorescein phosphoramidite-labeled duplexes IVa, IVb and IVc (Table 3.2). Even numbered lanes from 2 to 12 show the results of mechlorethamine incubation with duplex IVa fluorescein-labeled on the top strand (lane 2), duplex IVa fluorescein-labeled on the bottom strand (lane 4), duplex IVb fluorescein-labeled on the top strand (lane 6), duplex IVb fluorescein-labeled on the bottom strand (lane 8), duplex IVc fluorescein-labeled on the top strand (lane 10) and duplex IVc fluorescein-labeled on the bottom strand (lane 12). Odd numbered lanes from 1 to 11 are controls (no mechlorethamine) for the indicated duplexes. Bands are identified as X (crosslink), M (monoadduct), and S (unreacted single strands). Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. duplex VI VII T B T t +b B Figure 3.5. Autoradiograms of 20% DPAGE gels following incubation with lOOpM mechlorethamine of 5 '-fluorescein phosphoramidite-labeled duplexes VI and VII (Table 3.2). Bands are identified as X (crosslink), M (monoadduct), and S (unreacted single strands). A. Results of mechlorethamine incubation with duplex VI fluorescein-labeled on the top strand (lane 2) and duplex VI fluorescein-labeled on the bottom strand (lane 4). Odd numbered lanes are controls (no mechlorethamine). B. Results of mechlorethamine incubation with duplex VII fluorescein-labeled on the top strand (lane 2), duplex VII fluorescein-labeled on the bottom strand (lane 4) and duplex VII double fluorescein-labeled on the top and bottom strands. Lane 1 is a control (no mechlorethamine) for duplex VII fluorescein-labeled on the top strand. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 75 IVa IVb IVc duplex T B T B T B Figure 3.6. Autoradiogram of a 20% DPAGE gel following incubation with lOOpM mechlorethamine of duplexes IVa, IVb and IVc (Table 3.2) carrying both 5'-3 2 P- phosphate and 5'-fluorescein phosphoramidite labels. The gels in A and B are identical, but the bands in A are visualized via the 3 2 P label, whilst those in B are visualized via the fluorescein label. A. Even numbered lanes show the results of mechlorethamine incubation with duplex IVa P-labeled on the top strand and fluorescein-labeled on the bottom strand (lane 2), duplex IVa 3 2 P-labeled on the bottom strand and fluorescein-labeled on the top strand (lane 4), duplex IVb 3 2 P- labeled on the top strand and fluorescein-labeled on the bottom strand (lane 6), duplex IVb 3 2 P-labeled on the bottom strand and fluorescein-labeled on the top strand (lane 8), duplex IVc 3 2 P-labeled on the top strand and fluorescein-labeled on the bottom strand (lane 10) and duplex IVc 3 2 P-labeled on the bottom strand (and fluorescein-labeled on the top strand (lane 12). Odd numbered lanes from 1 to 11 are controls (no mechlorethamine) for the equivalent duplexes. B. Lanes 1 to 12 are as in A, but the gel is visualized via the fluorescein label, and the label location indicated (T or B) below the lanes refers to the strand carrying the fluorescein label. Bands are identified as X (crosslink), M (monoadduct), and S (unreacted single strands). Note the reversal in the mobility of the single-stranded, unreacted DNA in B, compared to the equivalent lane in A, which occurs because the opposite strand is being detected in B, compared to A. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 76 3.4 Discussion The analysis of complex conformers of d[CCG]n through initial mechlorethamine crosslinking of the C-C mismatched pairs will require identification of each crosslink band. Hence, an understanding of the electrophoretic mobility properties of these crosslinks is of importance, particularly because an initial work (Chapter II) suggested that these properties are anomalous in several respects. These include an apparent dependence of crosslinked duplex mobility on (i) the overall duplex stability, (ii) the position of the crosslink with respect to the duplex end, (iii) the position of the label on the duplex, and (iv) the nature of the label used. In the following discussion, each of these issues is addressed. It is apparent that, for duplexes carrying a single mechlorethamine C-C crosslink, those that include other C-C mismatches have a retarded mobility, compared to those with only Watson-Crick pairs. This apparent relationship of duplex stability with electrophoretic mobility is consistent with previous observations (Chapter II) on the effect of GC:AT content on the mobility of the mechlorethamine crosslinked duplex (Romero et al., 2001). It is also consistent with data reported for the mobility of psoralen-crosslinked duplexes, which have a decreased DPAGE mobility when destabilized by either inclusion of mismatched pairs, or by decreasing the overall GC content of the duplex (Kumaresan et al, 1992). Based on these data, it was hypothesized that the crosslinked duplex does not Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 77 denature entirely in a denaturing gel, and that a greater mobility is related to the maintenance of a part-duplex structure (Kumaresan et al., 1992). Consistent with this, it has also been shown that, for (uncrosslinked) duplexes of 200 base pairs, a higher G-C content leads to a greater mobility (Fischer and Lerman, 1979; Fischer and Lerman, 1983; Myers et al., 1987). The dependence of the mobility on the crosslink position may also be a function of duplex stability. For 19 base pair duplexes with centrally positioned (Qo- C2 9 ) crosslinks, fraying at each end would reduce the duplex content and, hence, the mobility, and this effect would not be influenced by the crosslink. In contrast, as the crosslink is positioned closer to one end, the fraying at the end proximal to the crosslink (the ‘short’ duplex end) may be reduced by the stabilizing effect of the crosslink, whilst fraying at the end distal to the crosslink would be similar to that in the duplex with the central crosslink. Hence, displacement of the crosslink towards the duplex end results in a more stable duplex conformation in the denaturing gel, and a greater electrophoretic mobility. This hypothesis can be used to explain the subtle differences in mobility between, for example, crosslinked duplexes Iy and Iz (Figure 3.2A), because duplex Iy has a higher GC content at the ‘short’ duplex end (Table 3.1) and hence forms a more stable duplex in this region. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 78 The mobility of the C7-C32 crosslinked duplex Ily confirms the importance of end-fraying in determining the duplex mobility. This crosslinked duplex has a similar mobility to duplexes Iy, Iz, IVb and Vb (all of which also have C7-C32 crosslinks), despite the presence of an additional C-C mismatched pair (C10-C29) in duplex Ily. However, this mismatched pair is sufficiently distant from the duplex end distal to the crosslink to influence the fraying at this end. This is in contrast to the C7-C32 crosslinked duplex IIx, in which the additional C-C mismatched pair (C13-C26) is closer to the duplex end that is distal to the crosslink, and presumably is able to increase the fraying of that end, and reduce the overall mobility of the duplex (contrast band b, lane 10, Figure 3.2A with band b, lane 6, Figure 3.2A). End-fraying effects may also explain the different mobility observed for identical crosslinked duplexes that differ only in the presence of a label on the ‘top’ or ‘bottom’ strand. For the 5 '-3 2 P-phosphate labeled mechlorethamine C-C crosslinked duplexes IVb and IVc (Figure 3.3A) and Vb (Figure 3.3B) the duplex carrying a label distal to the crosslink (on the bottom strand) has a greater electrophoretic mobility than the equivalent duplex with a label proximal to the crosslink (on the top strand). Continuing from the above hypothesis regarding end- fraying of the duplexes, the duplex end proximal to the crosslink may be somewhat destabilized by the presence of the 5 '-3 2 P-phosphate label (and additional negative charge), whilst the duplex end distal to the crosslink will be relatively unaffected Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 79 (because it is already frayed). Hence, the ‘top’ strand labeled species will have relatively less duplex character, compared to the ‘bottom’ strand labeled species, and therefore will have a reduced mobility in the denaturing gel. The effect of the 5'-fluorescein phosphoramidite label appears to be the opposite of the 3 2 P-phosphate label. Hence, for duplexes IVb (Figure 3.3A) and VII (Figure 3.5B), a fluorescein label proximal to the crosslink (on the top strand) enhances the mobility of the crosslinked duplex, compared to the same species having the label distal to the crosslink (on the bottom strand). Again following the end-fraying hypothesis, this perhaps suggests that the fluorescein moiety is able to further stabilize the ‘short end’ of the duplex, whilst having less of an effect on the already frayed end distal to the crosslink. The effect of the fluorescein label is also observed for the centrally crosslinked duplexes IVa and VI (which are of ‘reverse’ sequence, Table 3.2). For each crosslinked duplex, the mobility is enhanced when the fluorescein label is proximal to the GC-rich part of the duplex (contrast lanes 2 and 4 of Figure 3.4 and lanes 2 and 4 of Figure 4.5A). That the 5'-fluorescein phosphoramidite and 5 '-3 2 P-phosphate labels appear to have opposite effects indicate that the charge and hydrophobicity of the moiety used to end-label the individual strands have an influence on the stability of the duplex end, and consequently, the relative duplex mobility. Based on the theory that the 5 '-fluorescein phosphoramidite label has a stabilizing effect and the 5 '-3 2 P-phosphate label has a destabilizing effect, Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 80 the absence of the differential mobility within each duplex containing both labels as observed in Figure 3.6A and B can be explained by the offsetting of two counteracting effects (compare lanes 2 and 4, lanes 6 and 8, or lanes 10 and 12). It can be concluded that the electrophoretic mobility of mechlorethamine C-C crosslinked duplexes is determined by the stability of the crosslinked species, especially the stability at the duplex end. A tight and stable duplex end is the site that preferentially leads the crosslinked molecules migrating in the gel. Hence, increasing the stability of a duplex end (tight structure) by (i) increasing the GC content, (ii) having the crosslink position close to the duplex end, or (iii) introducing a label that has a stabilizing effect allows the molecules to migrate on the gels easily, and results in the faster electrophoretic mobility. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 81 CHAPTER IV Extrahelical Cytosine Bases in DNA Duplexes Containing d[GCC]n *d[GCC]n Repeats: Detection by a Mechlorethamine Crosslinking Reaction 4.1 Introduction Mismatched base pairs can be incorporated into DNA duplexes during replication and recombination. The repair of such potentially mutagenic lesions occurs with variable efficiency, depending on the nature of the mismatched pair (Kramer et al., 1984; Su et al., 1988; Gasc, 1989). The cytosine-cytosine (C-C) pair is an example of a poorly repaired mismatch (Kramer et al., 1984; Su et al., 1988; Gasc, 1989). The efficiency of repair can be correlated with the thermodynamic stability of the mismatched pair (Wemtges et al., 1986), and the C-C mismatch is amongst the least stable (Aboul-ela et al., 1985; Peyret et al., 1999). Mismatch recognition, and therefore the structure of the mismatched pair, is a key element in the repair process. An early study of the C-C mismatched pair proposed an intrahelical structure that could be stabilized by protonation (Brown et al., 1990). More recent solution state nuclear magnetic resonance (NMR) data also suggested an intrahelical C-C mismatched pair (Boulard et a l, 1997). However, the bases of a C-C Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 82 mismatched pair can also adopt ‘extrahelical’ locations in the minor groove of the duplex, in the so-called E-motif conformation (Gao et al., 1995). This structure is returned below. In the past decade, mismatched pairs have also been shown to be of significance in the formation of unusual DNA conformations (Sinden, 1999) associated with the so-called triplet repeat expansion diseases, or TREDS (Timchenko and Caskey, 1999; Jin and Warren, 2000; Bowater and wells, 2001). For example, both strands of the d[CGG]n »d[CCG]n triplet repeat sequence associated with Fragile X syndrome (Jin and Warren, 2000) form hairpin structures which include mismatched pairs (Gacy et a l, 1995; Chen et al., 1995; Mitas et al., 1995b; Nadel et al., 1995; Zheng et al., 1996; Mariappan et al., 1996b,1998; Yu et al., 1997; Usdin, 1998; Darlow and Leach, 1998a,1998b). The d[CCG]n hairpin has a C-C mismatch at every third base pair of the hairpin stem (Mariappan et al., 1996b; Darlow and Leach, 1998a,1998b; Mariappan at al., 1998; Zheng et al., 1996; Yu et al., 1997). These hairpins are conformationally flexible, and may contain C-C mismatched pairs in both intrahelical and extrahelical conformations. For further details, the reader is referred to two discussions of the d[CCG]n hairpin conformational flexibility (Darlow and Leach, 1998a, 1998b; Romero et al, 2001). Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 83 The discovery of the E-motif structure by Gao et al. (1995) provided the first evidence that the flexibility of mismatched pairs could lead to a major conformational change within a duplex sequence. The E-motif contains a central d[GCC]«d[GCC] fragment in which the mismatched cytosine bases are extrahelical. The thermodynamic driving force for the conformational change is provided by stacking of the guanine bases within the pseudo-dinucleotide d[GC]«d[GC] step that forms following unstacking of the mismatched bases (Figure 4.1). Based on the observation of a distorted d[CCG]is hairpin containing a d[GCC]n«d[GCC]n repeat stem, Yu et al. (1997) proposed that this hairpin might be partially in the form of an extended E-motif, in which the bases of multiple C-C mismatched pairs adopt extrahelical locations. The putative extended E-motif structure is shown schematically in Figure 4.1. As a corollary to these studies, it has been shown that the common nitrogen mustard, mechlorethamine (Figure 4.2A), can form a DNA interstrand crosslink (Figure 4.2B) at a C-C mismatched pair (Romero et al., 1999; Romero et al., 2001). Preliminary molecular modeling of the putative extended E-motif structure (Figure 4.1) suggested that the N3 atoms of proximal, extrahelical cytosine bases (for example, Cio and C3 2 in Figure 4.1) may be only about 5 A apart. Given this, it is rationalized that pairs of extrahelical cytosine bases might be susceptible to mechlorethamine crosslinking. In this chapter, it is shown that DNA duplexes Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 84 containing two and three contiguous d[GCC]*d[GCC] helical fragments can be crosslinked by mechlorethamine at all the formal C-C mismatched pairs, and between specific pairs of cytosine bases that are not formally paired in the duplex structure. These results provide evidence for an extended E-motif DNA (eE-DNA) conformation. 5 C • 6 13C O C; G * C C • G o G G • C C * G 7C o C G * C S' A B Figure 4.1. Schematic representation of the possible conformers of a d[GCC]3 ed[GCC] . 3 duplex fragment, showing molecules containing Watson-Crick pairs (hydrogen bonds represented by filled circles) and C-C mismatch pairs (open circles). The cytosine bases of the C-C mismatch pairs are labeled based on the numbering system of duplex III (Figure 4.2C), which contains the d[GCC]3 *d[GCC] 3 fragment. A. A duplex in which C7 -C3 2 , C 1 0 -C2 9 and C 1 3 -C2 6 are formal, intrahelical C-C mismatch pairs. B. A duplex containing extrahelical cytosine bases, in which C 1 0 -C3 2 and C 1 3 -C2 9 form extrahelical pseudo-pairs. 2 6 2 9 32 c j T a 13 r 10 C , / G 5' s c c « / * • c = / 32 c Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 85 Sugar Sugar c , S ' -CTCTCGCCGCCGCCGTATC GAGAGC GGCCGCGGCATAG- 5 ' „ S ' -CTCTCGCCGCCGCCGTATC GAGAGCCGCCGCGGCATAG-5' U » 5 ' -CTCTCGCCGCTGCCGTATC GAGAGCCGCCGCCGCATAG- S ' . y 5 ' -CTCTCCCGCCGCCGGTATC GAGAGGCCGCCGGCCATAG-S' Figure 4.2. A. Mechlorethamine. B. A representation of the DNA interstrand crosslink formed by mechlorethamine at a C-C mismatch pair, showing the probable connectivities of the mechlorethamine crosslink through the cytosine N3 atoms. C. Duplex sequences containing a single C-C mismatch pair (C 1 0 -C2 9 , duplex I), two C- C mismatch pairs (C7 -C3 2 and C1 0 -C2 9 , duplex II), three C-C mismatch pairs (C7 -C3 2 , C 1 0 -C2 9 and C 1 3 -C2 6 , duplex III), and two C-C mismatch pairs (C7 -C3 2 and C 1 0 -C2 9 , duplex IV). Cytosine bases present as formal C-C mismatch pairs are underlined. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 86 4.2 Materials and Methods Chemicals and reagents: Mechlorethamine and T4 polynucleotide kinase 3 9 were purchased from Sigma, [y- P]ATP was purchased from ICN. Oligodeoxyribo- nucleotides were synthesized on an Applied Biosystems Model 394 automated synthesizer, deprotected, and purified with a COP cartridge. Other reagents were at least analytical grade. 3 2 P 5 ’ -end Labeling o f DNA: Column purified synthetic DNA (2.5 pg, 0.5 nanomoles) was 5'-end labeled with [y-3 2 P]ATP (5 pi, 4500 Ci/mmol) by incubation in buffer (30mM Tris (pH 7.8), 10 mM MgCh, 5mM dithiothreitol) and 30 units of T4 polynucleotide kinase for 1 hour at 37°C. The reaction was stopped by addition of 3M sodium acetate (5.5pL, pH 5.2) and pre-chilled 95% ethanol (150 pL). Unincorporated [y-3 2 P]ATP was removed by precipitation in pre-chilled 95% ethanol (200pL) at -20°C overnight. The labeled DNA was lyophilized and resuspended in a 0.1M NaCl solution. Alkylation o f DNA: The unlabeled complementary strand (2.5 pg) was added to a 0.1M NaCl solution of the labeled oligodeoxyribonucleotide, heated to 65°-70°C and then slowly cooled to room temperature. It is noted that the unlabeled strand is in excess, given the less than 100% recovery of the labeled DNA. Following annealing Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 87 of the strands, a lpM duplex DNA solution containing 0.1M NaCl and lOmM Tris (pH 7.5) was incubated at 37°C with lOOpM mechlorethamine in a total volume of lOOpL, and for the times indicated. For each experiment, a fresh solution of lOOmM mechlorethamine was prepared in dimethyl sulfoxide (DMSO), rapidly diluted to lOmM and immediately added to the DNA solution. Following incubation, the reaction was terminated by addition of 3M sodium acetate (5.5pL, pH 5.2), tRNA (5mg/mL, 5 pL) and pre-chilled 95% ethanol (150pL), and precipitated in pre- chilled 95% ethanol (200pL) at -20°C, washed and lyophilized. The DNA was then dissolved in distilled water (2pL) and tracking dye (8pL, 80% formamide, ImM EDTA, 0.025% bromophenol blue and xylene cyanol). Detection and Quantification o f Crosslinked DNA: The samples were loaded onto a 20% denaturing polyacrylamide gel [29:1 acrylamide/bisacrylamide, 8M urea, 89mM Tris-borate (pH 8.5) and 2mM EDTA (TBE buffer), 0.4mm thick, 38 x 31 cm, 2500 V, 45 W] until the xylene cyanol marker had migrated for a specific distance (duplex 110 cm; duplex II and IV 22 cm; duplex III 28 cm). After the gel was exposed to X-ray film, the intensity of each band was quantified using Kodak Digital Science ID software (Kodak Scientific Imaging Systems). Determination o f the Crosslinking Site: The bands due to the crosslinked DNA were recovered from the gel using the crush-and-soak procedure. The DNA Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 88 was then precipitated with ethanol, washed, lyophilized, and resuspended in 10% aqueous piperidine in a total volume of lOOpL. To ensure complete cleavage of all alkylated bases, the crosslinked samples were heated for 1 hour at 90°C. For the control Maxam-Gilbert G reaction, the DNA was cleaved using 10% aqueous piperidine, in a total volume of lOOpL, for 20 minutes at 90°C. Different incubation times for the control and mechlorethamine-treated DNA were required, because a 20 minutes incubation of the crosslinked DNA resulted in insufficient cleavage at the crosslinked sites. All samples were lyophilized overnight, resuspended in 2 pL distilled water and 8pL tracking dye (80% formamide, ImM EDTA, 0.025% bromophenol blue and xylene cyanol), heated at 90°C for 2 min, chilled in an ice bath, and loaded onto a 20% denaturing polyacrylamide gel (29:1 acrylamide/bisacrylamide, 8M urea, 89mM Tris-borate (pH 8.5), 2mM EDTA (TBE buffer), 0.4mm thick, 38 x 31 cm, 2500 V, 45 W) until the xylene cyanol marker had migrated 10 cm. 4.3 Results 4.3.1 A DNA duplex containing two C-C mismatched pairs gives four crosslinked species with mechlorethamine Duplex II (Figure 4.2C), which contains two C-C mismatched pairs, was incubated with mechlorethamine and the products of the reaction were separated by Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 89 electrophoresis on a 20% polyacrylamide denaturing gel (Figure 4.3A). Separate reactions were carried out in which the duplex was 3 2 P-labeled on the ‘top’ strand (Figure 4.3A, lane 2) or the ‘bottom’ strand (Figure 4.3A, lane 4). In each case, four crosslinked species were formed corresponding to bands a, b, c and d (Figure 4.3A, lane 2) and bands e, f, g and h (Figure 4.3A, lane 4), respectively. The DNA from each of these bands was extracted from the gel and subjected to Maxam-Gilbert sequencing. The results for each band are shown in Figures 4.3B and 4.3C. Band a (Figure 4.3A, lane 2) corresponds to a duplex crosslinked through Cio (Figure 4.3B) and band e (Figure 4.3A, lane 4) corresponds to a duplex crosslinked through C2 9 (Figure 4.3C). Flence, the slowest moving crosslinked species is a duplex crosslinked at the formal mismatched pair C 1 0 -C2 9 (Figure 4.4A). Similarly, bands b and f (Figure 4.3A) are due (Figures 4.3B and 4.3C) to a duplex crosslinked at the formal mismatched pair C7 -C3 2 (Figure 4.4B). The origins of the other bands in Figure 4.2A are discussed below. It is noted that for both pairs of bands (a and e, b and f, Figure 4.2A) the electrophoretic mobility of the crosslinked duplex varies slightly depending on the position of the 3 2 P-label (on the ‘top’ or ‘bottom’ strand). Control experiments were also performed in which either the ‘top’ or ‘bottom’ strands of the duplex were incubated separately with mechlorethamine. No crosslink bands were evident in any of the control experiments, proving that the bands in Figure 4.3A are due to interstrand crosslinks (data not shown). Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 90 A x j M S ■ T B I3lt G a b c d lb# G e f g h 1 2 3 4 Cm. * 1^34 .r* bm im ^ 12: • R < * ' h • M 9 m c 7 r* ^ -mm_| X J p f j j i j & m " ■ ■ ' ■ Figure 4.3. A. Autoradiogram of a 20% DP AGE gel showing the products of incubation for 1 hour of mechlorethamine with duplex II (Figure 4.2C) carrying a 5'- end 3 2 P-label on the ‘top’ strand (lane 2) and ‘bottom’ strand (lane 4). Lanes 1 and 3 are controls (no mechlorethamine). Bands are identified as X (crosslink), M (monoadduct), and S (unreacted single strands). Each individual band due to a mechlorethamine-crosslinked duplex is labeled from a to h, for identification purposes in the sequencing gels. B. Autoradiogram of a 20% DP AGE gel showing the products of Maxam-Gilbert sequencing of the crosslink bands from Figure 4.3 A, lane 2, which correspond to crosslinked duplexes carrying a 5 '-end 3 2 P-label on the ‘top’ strand. The lane labeled G shows the results of a Maxam-Gilbert guanine sequencing reaction, and identifies the positions on the gel of fragments cleaved at four guanine bases in the ‘top’ strand. Lanes a, b, c and d refer to the bands from Figure 4.3A, and correspond to cleavage at Cio (lanes a and d) or C7 (lanes b and c). C. Autoradiogram of a 20% DP AGE gel showing the products of Maxam-Gilbert sequencing of the crosslink bands from Figure 4.3A, lane 4, which correspond to crosslinked duplexes carrying a 5'-end 3 2 P-label on the ‘bottom’ strand. The lane labeled G shows the results of a Maxam-Gilbert guanine sequencing of the ‘bottom’ strand. Lanes e, f, g and h refer to the bands from Figure 4.3A, and correspond to cleavage at C2 9 (lanes e and g) or C3 2 (lanes f and h). Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 91 a C * G « C * G a C • O « c * G26 mC * G?s .,C * G26 G • C G » C G • C C • G C * G C • G l 0C-M-C29 „ c o c m 10c - m - c 29 G • C G • C G • C C » G C • G C • G ,C O c 32 , c - m- c32 3C-M~C32 G • C G • C G • C S ' S ' 5 ' C » G jjC • g2S G ♦ C c * G 13C * G2« G • c C / g • G • C 10 C •G?3 2 / G • c ,C 5' “t c C * G G * C> ij C 1 H _ C j a 7j^C • G 2 9 G * C'\ 5' C S 2 Figure 4.4. Possible mechlorethamine-crosslinked species for duplex II (Figure 4.2C). The central 9 base pairs of the duplex are shown, and M indicates the location of a mechlorethamine crosslink. A. C10-C29 intrahelical crosslink. B. C7-C32 intrahelical crosslink. C. C10-C29 and C7-C32 double intrahelical crosslink. D. C10-C32 extrahelical crosslink in an extended E-motif DNA (eE-DNA) conformation with 5' foldback of the extrahelical cytosine bases. E. C7-C29 extrahelical crosslink in an eE- DNA conformation with 3 ' foldback of the extrahelical cytosine bases. This conformation was not observed under the conditions used. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 92 4.3.2 Extrahelical cytosine bases can be crosslinked by mechlorethamine: Evidence for an extended E-motif DNA (eE-DNA) conformation In the E-motif DNA structure (Gao et a l, 1995) the extrahelical cytosine bases in the d[GCC]«d[GCC] duplex fragment are positioned in the DNA minor groove, and folded back towards the 5'-end of their respective strands. If duplex II contained an extended E-motif (eE-DNA) structure with similar properties, a crosslink could conceivably form between Cio and C3 2 (Figure 4.4D). On the other hand, if the extrahelical cytosine bases fold back towards the 3'-end of their respective strands, then a C7 -C2 9 crosslink might be formed (Figure 4.4E). Band d (Figure 4.3A, lane 2) results from a crosslink formed through Cio (Figure 4.3B). Similarly, band h (Figure 4.3A, lane 4) results from a crosslink formed through C3 2 (Figure 4.3C). This result is consistent with crosslinking of the duplex at C 1 0 -C3 2 (Figure 4.4D). A similar analysis of the origin of band c (Figure 4.3 A, lane 2) and band g (Figure 4.3A, lane 4) suggests the presence of a duplex crosslinked through C7 -C2 9 (Figure 4.4E). However, it is also apparent that a duplex containing intrahelical C-C mismatched pairs and crosslinks at both C7 -C3 2 and C 1 0 -C2 9 (Figure 4.4C) would give identical sequencing data (through cleavage of the alkylated cytosine bases proximal to the P-label on each strand, C7 and C2 9 ). The identity of the species giving bands c and g is resolved in the following section. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 93 4.3.3 A kinetic analysis suggests that multiple mechlorethamine C-C crosslink formation can occur in a single duplex The origin of bands c and g (Figure 4.3A) for duplex II was determined by observing the formation of the crosslink bands as a function of time (Figure 4.5). Crosslink formation in duplex II at the formal mismatched C-C pairs, C 1 0 -C2 9 and C7 -C3 2 (bands a and b, respectively, Figure 4.5A), reaches a maximum of 14% (Cto- C2 9 ) and 12% (C7 -C3 2 ) of the total DNA after 80 minutes (Figure 4.5B). For time points beyond 80 minutes, there is apparent elimination of the species carrying either the C 1 0 -C2 9 or the C7 -C3 2 crosslink. It is concluded that this is due to the conversion of species with single crosslinks into a species having two crosslinks (at both C 1 0 -C2 9 and C7 -C3 2 , Figure 4.4C). Hence, band c (Figures 4.3A and 4.5A) and band g (Figure 4.3A) are due to a double crosslinked species (Figure 4.4C). The rate of formation (Figure 4.5B) of the species giving band c is consistent with this conclusion, and the amount of this species continues to rise as the single crosslink species are eliminated (Figure 4.5B). To confirm that the elimination of the single crosslink species in duplex II was due to their conversion to double crosslinked duplexes, and not just to degradation of the C 1 0 -C2 9 and C7 -C3 2 crosslinks, a kinetic analysis of crosslinking of duplex I, which contains only one C-C mismatched pair was performed (Figure 4.2C). Formation of the C 1 0 -C2 9 intrahelical crosslink in duplex I reaches about 28% of the total DNA after 100 minutes, beyond which no change is observed (Figure 4.6). Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 94 0 to 20 20 40 50 60 80 100 120 150 ISO 240 200 B 15 n c Q Sp 3s * 180 240 300 120 0 60 Time (minutes) Figure 4.5. Kinetics of mechlorethamine interstrand crosslink formation with duplex II (Figure 4.2C) for reaction times up to 5 hours. A. Autoradiogram of a 20% DP AGE gel following incubation of 5'-end ‘top’ strand 3 2 P-labeled duplex II with 100pM mechlorethamine. Lane 0 is a control (no mechlorethamine) and all other lanes show the products of incubation for the time (in minutes) indicated above the lane. Bands are identified as X (crosslink), M (monoadduct), and S (unreacted single strands), and individual crosslink bands are labeled a to d, consistent with Figure 4.3A (lane 2). B. Quantification of the autoradiogram showing the time course of crosslink formation for band a (C10-C29, intrahelical) (filled squares), band b (C7-C32, intrahelical) (filled triangles), band c (C7-C32 and C10-C29, double intrahelical) (open circles) and band d (C10-C32, extrahelical) (open squares). Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 95 0 10 2(1 3(1 4(1 50 Wt SO 100 120150 ISO 24il.VtO B o o 30 25 20 15 10 5 0 180 240 300 120 0 60 Time {minutes} Figure 4.6. Kinetics of mechlorethamine interstrand crosslink formation with duplex I (Figure 4.2C) for reaction times up to 5 hours. A. Autoradiogram of a 20% DP AGE gel following incubation of 5'-end ‘top’ strand 3 2 P-labeled duplex I with lOOpM mechlorethamine. Lane 0 is a control (no mechlorethamine) and all other lanes show the products of incubation for the time (in minutes) indicated above the lane. Bands are identified as X (crosslink), M (monoadduct), and S (unreacted single strands). B. Quantification of the autoradiogram showing the time course of crosslink formation of the C 1 0 -C2 9 intrahelical crosslink. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 96 4.3.4 A duplex containing three C-C m ism atched pairs gives three intrahelical and tw o extrahelical C-C crosslinked species w ith m echloretham ine To confirm the conclusions drawn from duplex II, the mechlorethamine crosslinking reactions of duplex III, which contains three C-C mismatched pairs (Figure 4.2C) was examined. Separate reactions were carried out in which duplex III was P-labeled on the ‘top’ strand (Figure 4.7A, lane 2) or the ‘bottom’ strand (Figure 4.7A, lane 4). In each experiment, five crosslinked species were apparent on the denaturing gel, corresponding to bands a, b, c, d and e (Figure 4.7A, lane 2) and bands f, g, h, i and j (Figure 4.7A, lane 4), respectively. The sequencing of each of these bands is shown in Figures 4.7B and 4.7C, and the structures of the five crosslinked species that were identified are shown in Figure 4.8. An analysis of the sequencing gels shows that the crosslinked species corresponding to bands a and f (Figure 4.7A) is that containing a C10-C29 intrahelical crosslink (Figure 4.8A). Similarly, bands b and g are due to a C 1 3 -C2 6 intrahelical crosslink (Figure 4.8B) and bands c and h correspond to a C7-C32 intrahelical crosslink (Figure 4.8C). The two pairs of faster moving bands (d and i, e and j, Figure 4.7A) are due to crosslinking of eE-DNA conformers containing extrahelical cytosine bases folded towards the 5'- end. Hence, bands d and i result from a C10-C32 extrahelical crosslink (Figure 4.8D) and bands e and j correspond to a C13-C29 extrahelical crosslink (Figure 4.8E). Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 97 A x M ► S T B W % D G 'a h c d e Q | G f g h i j 1 2 3 4 r . bate 8 - * b w ^*15 « * d * S e J Cw • 4 '•*31 • M o • W28 C 7 * ei U ^ l | m ^'25 & Figure 4.7. A. Autoradiogram of a 20% DP AGE gel showing the products of incubation for 1 hour of mechlorethamine with duplex III (Figure 4.2C) carrying a 5'-end 3 2 P-label on the ‘top’ strand (lane 2) and ‘bottom’ strand (lane 4). Lanes 1 and 3 are controls (no mechlorethamine). Bands are identified as X (crosslink), M (monoadduct), and S (unreacted single strands). Each individual band due to a mechlorethamine-crosslinked duplex is labeled from a to j, for identification purposes in the sequencing gels. B. Autoradiogram of a 20% DP AGE gel showing the products of Maxam-Gilbert sequencing of the crosslink bands from Figure 4.7A, lane 2, which correspond to crosslinked duplexes carrying a 5 '-end 3 2 P-label on the ‘top’ strand. The lane labeled G shows the results of a Maxam-Gilbert guanine sequencing reaction, and identifies the positions of fragments cleaved at four guanine bases in the ‘top’ strand. Lanes a, b, c, d and e refer to bands from Figure 4.7A, and correspond to cleavage at C7 (lane c), Cio (lanes a and d) or C 1 3 (lanes b and e). C. Autoradiogram of a 20% DP AGE gel showing the products of Maxam-Gilbert sequencing of the crosslink bands from Figure 4.7A, lane 4, which correspond to crosslinked duplexes carrying a 5'-end 3 2 P-label on the ‘bottom’ strand. The lane labeled G shows the results of a Maxam-Gilbert guanine sequencing of the ‘bottom’ strand. Lanes f, g, h, i and j refer to bands from Figure 4.7A, and correspond to cleavage at C26 (lane g), C29 (lanes f and j) or C32 (lanes h and i). Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 98 D A C c * G c • G c • G O *-2 6 13*-" -M-c26 0 '-'26 G * C 6 • C G * C C • G C * G C * G 10c - m- c „ 1 0 C 0 C29 0 c 29 G * C G • € G • c c * G C • G c • 0 , c O C32 ? c 0 £32 ,C--M-c3 2 G * c G • C G * c 5 ' S ' S ' 1 3 v c f s : c /a i C • G C »G s£» gy ? * 6 C 4 ^ * 2 9 c Qp32 C -2 6 15 C /'G * ioc c * *^G * 7C 5' Gjp9 C G^ ’ c £ ■ 3 2 Figure 4.8. Mechlorethamine-crosslinked species for duplex III (Figure 4.2C). The central 9 base pairs of the duplex are shown, and M indicates the location of a mechlorethamine crosslink. A. C 1 0 -C2 9 intrahelical crosslink. B. C 1 3 -C2 6 intrahelical crosslink. C. C7-C32 intrahelical crosslink. D. C10-C32 extrahelical crosslink. E. C13- C29 extrahelical crosslink. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 99 4.3.5 Double crosslinks can also form in a duplex containing three C-C mismatched pairs To confirm the results for duplex HI, a similar kinetic analysis was performed to those described for duplexes I and II. These data are shown in Figure 4.9. In addition to the five crosslinked species identified above, bands due to further species develop after about 30 minutes (bands labeled XX in Figure 4.9A). Specific identification of these bands has not been attempted, but presumably they correspond to the three different ways of forming two intrahelical C-C crosslinks in duplex III. It is noted that the decrease in the intensity of the single crosslink bands, as these species are converted to the double crosslink species, is not as pronounced for duplex III (Figure 4.9B), compared to duplex II (Figure 4.5B). This is probably due to the greater number of bands present for duplex III, and the correspondingly lower intensity. However, there does appear to be a small, but consistent, decrease in the intensity of the single crosslink species for duplex III after 1 hour (Figure 4.9B). Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 100 M-4 s — * 7 i C c C O W 5 4 O 3 2 1 0 0 120 240 Time (minutes) Figure 4.9. Kinetics of mechlorethamine interstrand crosslink formation with duplex III (Figure 3.2C) for reaction times up to 4 hours. A. Autoradiogram of a 20% DP AGE gel following incubation of 5 '-end ‘top’ strand 3 2 P-labeled duplex III with mechlorethamine. Lane 0 is a control (no mechlorethamine) and all other lanes show the products of incubation for the time (in minutes) indicated above the lane. Bands are identified as X (single crosslinks), XX (double crosslinks), M (monoadduct), and S (unreacted single strands), and individual crosslink bands are labeled a to e, consistent with Figure 4.7A (lane 2). B. Quantification of the autoradiogram showing the time course of crosslink formation for band a (C 1 0 -C2 9 , intrahelical) (filled circles), band b (C 1 3 -C2 6 , intrahelical) (filled squares), band c (C7 -C3 2 , intrahelical) (filled triangles), band d (C 1 0 -C3 2 , extrahelical) (open squares), band e (C 1 3 -C2 9 , extrahelical, data obscured by the band d data) (open triangles), and bands representing three possible double, intrahelical crosslink species (crosses). Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 101 4.3.6 A duplex containing two contiguous d[CCG]*d[CCG] repeats does not undergo crosslinking of cytosine bases that are not formally paired Duplex IV contains two d[CCG]»d[CCG] repeat duplex fragments, and two formal C-C mismatched pairs positioned similarly to those in duplex II (Figure 4.2C). Incubation of duplex IV with mechlorethamine gave only two crosslinked species for reaction times up to 6 hours (Figure 4.10). These bands correspond to intrahelical crosslinks at C 1 0 -C2 9 and C7 -C3 2 . No evidence of crosslinking between extrahelical cytosine bases was obtained, suggesting that the eE-DNA conformation cannot form in duplex IV. 4.3.7 Quantification of the mechlorethamine C-C crosslinking reactions Quantitative data derived for mechlorethamine crosslinking of duplexes I, II, III and IV are shown in Table 4.1. The data are shown as the amount of crosslinked DNA, expressed as a percentage of the total DNA, after four hours reaction time (by which time the mechlorethamine has either reacted with the DNA, or been hydrolyzed). For duplex I, the 28% crosslink formation for the intrahelical crosslink is consistent with previous reports of this reaction (Romero et al., 1999; Romero et al., 2001). For duplex II 31% of the DNA is crosslinked through intrahelical C-C mismatched pairs (including duplexes that carry two crosslinks). A further 8% of the total DNA is crosslinked through the extrahelical C10-C32 pair, giving an Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 102 approximate ratio of intrahelical: extrahelical crosslinks of 4:1. A similar analysis of the duplex III crosslinks gives a ratio of intrahelical : extrahelical crosslinks close to 2:1 (Table 4.1). 0 I I I 2 0 3 0 4 0 5 0 6 0 fid 1 0 0 1 2 0 15(1 1 8 0 2 4 0 5 6 0 • 1*1 20 15 10 5 0 60 120 180 240 300 360 0 Time (minutes) Figure 4.10. Kinetics of mechlorethamine interstrand crosslink formation with duplex IV (Figure 4.2C) for reaction times up to 6 hours. A. Autoradiogram of a 20% DP AGE gel following incubation of 5'-end ‘top’ strand 3 2 P-labeled duplex IV with mechlorethamine. Lane 0 is a control (no mechlorethamine) and all other lanes show the products of incubation for the time (in minutes) indicated above the lane. Bands are identified as X (crosslink), M (monoadduct) and S (unreacted single strands). B. Quantification of the autoradiogram showing the time course of crosslink formation for the C10-C29 intrahelical crosslink (filled squares) and the C7-C32 helical crosslink (filled circles). Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 103 Table 4.1. Quantification of mechlorethamine crosslinking of duplexes containing intrahelical and extrahelical C-C crosslink sites. Duplex Crosslink Position Crosslink Location Amount of Crosslink1 Total Crosslink1 5 Intra: Extra Ratio0 I i n o ! t o ! Intrahelical 28 28 - H C1 0-C 2 9 C7 -C3 2 C1 0-C 2 9 and C 7-C 3 2 C 1 0 -C3 2 Intrahelical Intrahelical Double Extrahelical 1 1 10 10 8 39 31 : 8 III C1 0-C 2 9 C1 3 -C2 6 c7 -c3 2 C 1 0 -C3 2 C 1 3 -C2 9 Three speciesd Intrahelical Intrahelical Intrahelical Extrahelical Extrahelical Double 5 6 6 5 5 3 x 2 33 23 : 10 IV C10-C29 C7 -C3 2 Intrahelical Intrahelical 1 8 16 34 34 : 0 a The amount of crosslinked DNA for each species, expressed as a percentage of the total DNA after 4 hours reaction time. b The total amount of crosslinked DNA for each duplex after 4 hours reaction time. c The ratio of intrahelical : extrahelical crosslinked species after 4 hours of reaction time, expressed as a percentage of the total DNA. d Species containing two intrahelical crosslinks (Figure 4.9A) were not sequenced for duplex III. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 104 4.4 Discussion Yu et al (1997) have proposed previously that multiple extrahelical cytosine bases might occur in d[GCC]n»d[GCC]n repeat duplex fragments, but they were unable to obtain direct evidence of an extended E-motif DNA (eE-DNA) conformation. This chapter has shown that pairs of cytosine bases in different formal C-C mismatched pairs, and located in a 1,4 interstrand relationship, can be crosslinked by mechlorethamine. In a B-DNA duplex the N3 atoms (through which it is believed the crosslink forms) of these cytosines are about 12A apart. Hence, if the cytosine bases remain intrahelical, this distance appears to be too large to allow mechlorethamine crosslinking, even if DNA bending occurs, as in the 1,3 G-G crosslink formed by mechlorethamine (Rink et al., 1993). It is possible that initial mechlorethamine monoadduct formation could induce an extrahelical cytosine conformation. However, this would still leave a significant distance between the monoadduct and the target (intrahelical) cytosine for completion of the crosslink, and it is believed that such a mechanism of crosslink formation is unlikely. It has also been considered the possibility of slippage of the DNA to bring different cytosine bases into proximity, but this seems unlikely, given the required disruption at the Watson-Crick paired duplex termini. It is also noted that, under the reaction conditions used, a significant amount of the 1,3 G-G crosslinked species was not expected (Romero et al., 2001), although it is possible that some of the weaker bands visible at longer incubation times may be due to such species. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 105 Given the above, it can be concluded that the mechlorethamine crosslink formed between cytosine bases that are not formally paired occurs because the bases are extrahelical. It can be further concluded that these bases are positioned similarly to those observed in E-motif DNA (Gao et al., 1995). The extrahelical cytosine bases (of a single C-C pair) in the E-motif are folded towards the 5 '-end of their respective strands (Gao et al., 1995). This positions the bases adjacent to the preceding d[CG] dinucleotide step in a deepened minor groove. These crosslinking data suggest that a similar arrangement occurs in extended E-motif DNA (eE-DNA), and that two extrahelical cytosine bases can form a ‘pseudo-pair’ in the minor groove. Crosslinks form between cytosine bases that would be proximal in 5'-end foldback conformations (C10-C32 in duplex II, and C10-C32 and C13-C29 in duplex III) (Figure 4.4 and 4.8). They are not observed for the equivalent pseudo-pairs (C7-C29 in duplex II, and C7-C29 and C10-C26 in duplex III) (Figure 4.4 and 4.8) that might be expected in a 3 '-end foldback conformation. Duplexes II and IV contain identically positioned formal C-C pairs, but the eE-DNA conformation is only adopted by duplex II. This may be because the transition to the extrahelical conformation in duplex II results in the formation of a pseudo-d[GC]»d[GC] step, which is energetically favorable because of effective stacking of the guanine bases (Gao et al., 1995). The equivalent transition in duplex IV would result in formation of a pseudo-d[CG]®d[CG] step. Presumably the Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 106 stacking interaction in this step is insufficient to drive the transition, and the eE- DNA conformation cannot form. It is also noted that there is no double, intrahelical crosslink species formed for duplex IV. This may be due to subtle differences in the duplex geometry following the initial crosslink formation. N.M.R. spectra of d[CCG]n hairpins suggest a dynamic equilibrium between conformers containing intrahelical and extrahelical cytosine bases (Zheng et al., 1996). A similar equilibrium appears to be present in the d[GCC]n»d[GCC]n duplex fragments, and it is apparent that there is a significant amount of eE-DNA present. The ratio of crosslinks (Table 4.1) does not necessarily reflect the ratio of intrahelical : extrahelical cytosine bases prior to crosslinking, because the rate of formation of the crosslink species may be different. However, it is of note that the intrahelical : extrahelical crosslink ratio is of the order of 4:1 in the d[GCC]2 *d[GCC] 2 fragment and about 2:1 in the d[GCC]3 *d[GCC] 3 fragment. This perhaps suggests greater stability of the extrahelical conformation with an increased number of repeats. It will be of interest to determine if extended runs of d[GCC]n»d[GCC]n repeats, and perhaps other sequences with appropriately spaced C-C mismatched pairs, adopt even more stable eE-DNA conformations. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 107 CHAPTER V Polyamine Inhibition of Mechlorethamine Cytosine-Cytosine Crosslinking Reaction with a DNA Duplex Containing a d[GCC]2«d[GCC]2 Fragment 5.1 Introduction Fragile X syndrome is characterized by expansions of DNA triplet repeat sequence d[CGG]n»d[CCG]n. In Fragile X patients, all the cytosine bases of the d[CG]«d[CG] dinucleotide steps within the d[CGG]»d[CCG] repeat region are methylated (Homstra et al., 1993). The single-stranded d[CCG]n probably forms intrastrand hairpin conformations with different stem alignments including some in an eE-DNA conformation as discussed in Chapter I and 4 (Figure 5.1) (Yu et al., 1997; Romero et al., 2001; Rojsitthisak et al., 2001). It is suggested that d[CCG]n hairpins are effective substrates for cytosine 5-methyltransferases (Chen et al., 1995) and that these enzymes require the target cytosine to be extrahelical (Klimasauskas et al., 1995). It is possible that the extrahelical cytosine bases may cause eE-DNA to act as a methyltransferase ‘sink,’ and cause the hypermethylation of the proximal promoter region of the FMR1 gene (without necessarily methylating the extrahelical cytosine itself). Alternatively, the presence of multiple d[CG]»d[CG] steps (Figure Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 108 5.IB) in the eE-DNA ‘core’ helix may provide for an effective methyltransferase substrate. For hairpin stems including d[GC]»d[GC] steps (Figure 5.1A(left)) the methylated cytosine base is part of a C-C pair, and this may facilitate the methylation reaction by making it easier for the extrahelical cytosine conformation to form (Chen et al., 1995). Although it was found in Chapter IV that the cytosine bases of the C-C mismatched pair in this alignment do not adopt extrahelical conformations in a d[CCGCCG]®d[CCGCCG] fragment, it is possible that extrahelical cytosine bases might occur in longer repeats of this alignment (Rojsitthisak et al., 2001). It has been shown that 5-azadeoxycytidine, a methyltransferase inhibitor, can reverse promoter methylation and increase the level of FMR1 gene expression (Chiurazzi et al., 1998), providing the first possible therapeutic approach to Fragile X syndrome. If extrahelical cytosine bases are important in causing promoter hypermethylation, then molecules designed to influence the equilibrium between the intrahelical and extrahelical bases may also have potential as Fragile X therapeutic agents. Figure 5.1. Schematic representation of the possible conformers of d[CCG]n, showing molecules containing Watson-Crick pairs (•) and C-C mismatched pairs (o). A. Alignments of d[CCG]n hairpin stems. B. Intrahelical and extrahelical C-C mismatched pairs within the hairpin stem. c o c c • G G • C 5' C G • C C o C C • G C • G C o C G • C C • G C o C G • C C • G C o C G • C 5' _ C * O ’ A B Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 109 Polyamines such as spermine, spermidine and putrescine (Figure 5.2) are cellular components with multiple functions in cell proliferation and differentiation. Under physiological pH conditions, polyamines are polycationic and hence they interact with anionic macromolecules such as DNA, RNA, proteins, and phospholipids (Marton and Pegg, 1995). Anionic phosphates of DNA are among the primary targets for charge neutralization by intracellular cations. Polyamines bind to DNA and neutralize these charges. Hydrogen bonding and hydrophobic interaction are also involved in the binding of polyamines to DNA (Tabor and Tabor, 1976; Tabor and Tabor, 1984; Panagiotidis et al., 1995). Figure 5.2. Structures of polyamines. A. Spermine B. Spermidine and C. Putrescine. It has been shown that polyamines have various effects on DNA structure. For instance, they can stabilize duplex DNA and a variety of unusual DNA conformations such as triplex DNA, the left-handed Z-DNA and the right-handed A- DNA (Hampel et al., 1991; Garriga et al., 1993; Musso and Van Dyke, 1995; Hasan .+ B h3 n+ \ ^ ^ h2 »+ C h 3 n+ Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 110 et al., 1995; Howell et al., 1996; Tippin et al., 1997; Musso et al., 1997). In addition, polyamines promote the transition from B to Z forms (Hasan et al., 1995; Howell et al., 1996). Polyamine binding also induces the condensation and aggregation of DNA, nucleosome and chromatin (Behe and Felsenfeld, 1981; Hougaard et al., 1987; Smirnov et al., 1988; Basu et al., 1990; Flock et al., 1996; Raspaud et al., 1999). Since polyamines can affect different DNA structures, they may have potential to influence the equilibrium between the intrahelical and extrahelical cytosine bases. The finding that mechlorethamine can crosslink both intrahelical and extrahelical cytosine bases suggests that mechlorethamine C-C crosslinking reaction can be used as a tool to test whether or not the designed molecules can influence the equilibrium between the intrahelical and extrahelical bases. To this end, it was shown in Chapter IV that duplex d[CTCTCGCCGCCGCCGTATC]«d[GATACG- GCGCCGCCGAGAG] containing two cytosine mismatched pairs within two d[GCC]«d[GCC] helical fragments could adopt structures containing intrahelical and extrahelical cytosine bases, and these structures could be detected by chemically crosslinking with mechlorethamine. It was shown that four mechlorethamine C-C crosslinks of this duplex are a C10-C29 intrahelical crosslink, a C7-C32 intrahelical crosslink, a C10-C29 and C7-C32 double intrahelical crosslink, and a C10-C32 extrahelical crosslink (Figure 5.3). In this chapter, the mechlorethamine C-C Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. I l l crosslinking reaction is used to investigate the effects of spermine in influencing the equilibrium between intrahelical and extrahelical cytosine bases using the above duplex as a model. The results show that spermine inhibited both crosslink formations at intrahelical and extrahelical cytosine bases, suggesting the lack of its influence on the equilibrium between the intrahelical and extrahelical bases. This implies that spermine may not be able to stabilize the intrahelical cytosine bases, nor specifically bind to the extrahelical cytosine bases and push them back into the intrahelical position. A c * G l 3C • g 26 G • C C • G i 0C -M -C 29 G • C C • G 7C o C32 G • C 5 ' Figure 5.3. M e c h lo re th a m in e -c ro s s lin k e d sp e c ie s fo r d u p le x d [C T C T C G C C G C C - G C C G T A T C ].d [G A T A C G G C G C C G C C G A G A G ]. T h e c e n tra l 9 b a s e p a irs o f th e d u p le x a re s h o w n , a n d M in d ic a te s th e lo c a tio n o f a m e c h lo re th a m in e c ro sslin k . A. C10-C29 in tra h e lic a l c ro sslin k . B. C7-C32 in tra h e lic a l c ro sslin k . C. C10- C29 a n d C7-C32 d o u b le in tra h e lic a l c ro s s lin k . D. C10-C32 e x tra h e lic a l c ro s s lin k in an e x te n d e d E - m o tif D N A (e E -D N A ) c o n fo rm a tio n w ith 5 ' fo ld b a c k o f th e e x tra h e lic a l c y to sin e b a se s. B 13 c c G C 1 0 C G C C- G 5 ' • c c 3* " G C 2 6 c G 10c - m - c 29 G C C G 7c - m - c 32 G • C 5 ' D c ,c G G, 13 26 G • C C • G ) r29 £ g • c - M - A 5 ' Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 112 5.2 M aterials and Methods Chemicals and reagents: Mechlorethamine, Spermine Tetrahydrochloride and T4 polynucleotide kinase were purchased from Sigma. [y-3 2 P]ATP was purchased from ICN. Oligodeoxyribonucleotides were synthesized on an Applied Biosystems Model 394 automated synthesizer, deprotected, and purified with a COP cartridge. Other reagents were at least analytical grade. 3 2 P 5 '-end Labeling o f DNA: Column purified synthetic DNA (2.5 pg, 0.5 nanomoles) was 5'-end labeled with [y-3 2 P]ATP (5pi, 4500 Ci/mmol) by incubation in buffer (30mM Tris (pH 7.8), 10 mM MgCb, 5mM dithiothreitol) and 30 units of T4 polynucleotide kinase for 1 hour at 37°C. The reaction was stopped by addition of 3M sodium acetate (5.5pL, pH 5.2) and pre-chilled 95% ethanol (150 pL). 'X ') Unincorporated [y- P] ATP was removed by precipitation in pre-chilled 95% ethanol (200pL) at -20°C overnight. The labeled DNA was lyophilized and resuspended in a 0.1M NaCl solution. Annealing o f DNA. The unlabeled complementary strand (2.5 pg) was added to a 0.1M NaCl solution of the labeled oligodeoxyribonucleotide, heated to 65°-70°C and then slowly cooled to room temperature. It is noted that the unlabeled strand is in excess, given the less than 100% recovery of the labeled DNA. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 113 Alkylation o f DNA: lpM duplex DNA solution containing 0.1M NaCl and lOmM Tris (pH 7.5) was incubated with 0, 0.5, 5, 50, 500 or 5000 pM spermine at 37°C for 1 hour and subsequently with lOOpM mechlorethamine for 2 hours in a total volume of lOOpL. For each experiment, a fresh solution of lOOmM mechlorethamine was prepared in dimethyl sulfoxide (DMSO), rapidly diluted to lOmM and immediately added to the DNA solution. Following incubation, the reaction was terminated by addition of 3M sodium acetate (5.5pL, pH 5.2), tRNA (5mg/mL, 5 pL) and pre-chilled 95% ethanol (150pL), and precipitated in pre- chilled 95% ethanol (200pL) at -20°C, washed and lyophilized. The DNA was then dissolved in distilled water (2pL) and tracking dye (8pL, 80% formamide, ImM EDTA, 0.025% bromophenol blue and xylene cyanol). Detection and Quantification o f Crosslinked DNA: The samples were loaded onto a 20% denaturing polyacrylamide gel (29:1 acrylamide/bisacrylamide, 8M urea, 89mM Tris-borate (pH 8.5) and 2mM EDTA (TBE buffer), 0.4mm thick, 38 x 31 cm, 2500 V, 45 W) until the xylene cyanol marker had migrated 22 cm. After the gel was exposed to X-ray film, the intensity of each band was quantified using Kodak Digital Science ID software (Kodak Scientific Imaging Systems). For a given spermine concentration, the intensity of the crosslink band was calculated as a fraction of the total DNA in the lane. These numbers for different spermine Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 114 concentrations were then related to each other using a scale based on 100% crosslinking at zero spermine concentration. 5.3 Results 5.3.1 The mechlorethamine C-C crosslinks are inhibited by spermine Duplex d[CTCTCGCCGCCGCCGTATC].d[GATACGGCGCCGCCGAGA- G] was equilibrated with serially diluted concentration of spermine followed by incubation with mechlorethamine. The four resultant crosslinked duplexes, which are the C10-C29 intrahelical crosslink (band a), C7-C32 intrahelical crosslink (band b), C10-C29 and C7-C32 double intrahelical crosslink (band c), and C10-C32 extrahelical crosslink (band d), were separated on a polyacrylamide gel from the unreacted single-stranded DNA and DNA monoadducts (Figure 5.4) and quantified by densitometry. For each spermine concentration, the intensity of the individual crosslink band was expressed as a percentage of the total DNA in the lane on the gel (Table 5.1). The total amount of crosslinked DNA in the absence of spermine is about 39% (Table 5.1), which is consistent with the findings of Chapter IV. A spermine concentration required to inhibit 50% the total mechlorethamine C-C crosslinks is about 450 pM (Table 5.1, Figure 5.5). Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 115 % X > P - > M — > S -> pM Spermine 1 » c d imii X 0 0.5 5 50 so o 5000 Figure 5.4. Autoradiogram of a 20% DP AGE gel showing spermine inhibition of mechlorethamine interstrand crosslinks for duplex d[CTCTCGCCGCCGCC- GTATC]. d[GATACGGCGCCGCCGAGAG]. The gel shows electrophoresis of the products following incubation of 5 -end ‘top’ strand P-label duplex with 100 pM mechlorethamine. Prior to this, the duplex had been pre-incubated with spermine at the concentrations indicated for each lane. Lane X is a control lane with no mechlorethamine and no spermine. Bands are identified as X (crosslink), P (spermine concentration-dependent band), M (monoadduct), and S (unreacted single strands). Individual crosslink bands are labeled a to d, corresponding to C10-C29, intrahelical (band a), C7-C32, intrahelical (band b), C7-C32 and C10-C29 double intrahelical (band c), and C10-C32, extrahelical (band d). 5.3.2 The spermine concentrations required to inhibit mechlorethamine cross- linking at intrahelical and extrahelical cytosine mismatched bases are similar The spermine concentrations required to inhibit 50% of the intrahelical crosslinks (C10-C29, C7-C32, and double crosslink) and extrahelical crosslink (C10- C32) are about 450 pM, in each case (Table 5.2, Figure 5.6). Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 116 Table 5.1. Spermine inhibition of mechlorethamine crosslinking of duplex d[CTCT- CGCCGCCGCCGTATC].d[GATACGGCGCCGCCGAGAG]. [Spermine] (pM) % Crosslink3 Intrahelical Extrahelical Total C10-C29 (Single) C7-C32 (Single) C10-C29 and C7-C32 (Double) Total Q0-C32 (Single) 0 11.7 9.3 8.7 29.6 9.2 38.7 0.5 11.6 6.5 8.4 29.4 9.1 38.5 5 11.2 9.0 7.4 27.5 8.3 35.9 50 11.1 8.5 6.4 26.0 8.2 34.2 500 9.1 5.8 4.0 18.8 5.7 24.5 5000 5.8 2.7 1.0 9.6 2.4 11.9 a The amount of crosslinked DNA for each species, expressed as a percentage of the total DNA. 40 30 20 10 0 0 0.5 5 50 500 5000 [Sperm ine] (jj,M) Figure 5.5. Spermine inhibition of mechlorethamine crosslinking of duplex d[CTCTCGCCGCCGCCGTATC].d[GATACGGCGCCGCCGAGAG], showing a decrease of total crosslinks as an increased amount of spermine. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 117 Table 5.2. Comparison of spermine inhibition of mechlorethamine crosslinking between intrahelical and extrahelical C-C crosslinking sites of duplex d[CTCT- CGCCGCCGCCGTATC].d[GATACGGCGCCGCCGAGAG]. [Spermine] 0*M) % Crosslink Intrahelical Extrahelical Total 0 100 100 100 0.5 99 99 99 5 93 90 93 50 88 89 88 500 64 62 63 5000 32 26 31 a The amount of crosslinked DNA for each species, expressed as a percentage of the total DNA (as shown in Table 5.1) and then related to each other using a scale based on 100% crosslinking at zero spermine concentration. 100 ■ * 80 w 60 M E 40 o 20 500 5000 5 50 0 0.5 [Spermine] (pM) Figure 5.6. Comparison of spermine inhibition of mechlorethamine C-C crosslinking of duplex d[CTCTCGCCGCCGCCGTATC].d[GATACGGCGCCGCCGAGAG], showing intrahelical crosslink (x), extrahelical crosslink (O) and total crosslink (♦). Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 118 5.4 Discussion It is apparent from the above results that the mechlorethamine C-C crosslinking reaction is inhibited at a high concentration of spermine. The results suggest that spermine may not specifically bind to the minor groove of this duplex. The interpretation of these results is based on the previous report that mechlorethamine C-C crosslinking reaction occurs in the minor groove of the DNA (Romero et al., 1999). It is possible that at low spermine concentration, spermine binds to either the GC-rich major groove or the phosphate backbone of the DNA duplex. At high spermine concentration, an excess spermine may eventually surround the DNA and block the crosslinking sites at the minor groove of the duplex, leading to the inhibition of mechlorethamine crosslink formation. This explanation is consistent with previous findings that spermine prefers to bind to AT-rich minor grooves and GC-rich major grooves. (Zakrzewska and Pullman, 1986; Marquet and Houssier, 1988; Feuerstein et al., 1989,1990; Haworth et al., 1991; Schmid and Behr, 1991; Yuki et al., 1996; Shamma and Haworth, 1999). The suggestion of non specific modes of polyamines binding to the phosphate backbone of DNA also support the above explanation (Wemmer et al., 1985; Padmanabhan et al., 1991). It is of note that there may be other possible explanations for these inhibition data. One such explanation could be the DNA condensation, aggregation, or precipitation induced by spermine (Gosule and Schellman, 1976,1978; Manning, Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 119 1978; Record et al., 1978; Wilson and Bloomfield, 1979; Widom and Baldwin, 1980; Thomas and Bloomfield, 1983). These phenomena can result in lowering the available DNA duplex for the crosslinking reaction, leading to a decrease of the crosslink formation. The DNA aggregation and precipitation are unlikely to happen in these studies. This is because the aggregation and precipitation occur at high DNA concentration, and the DNA concentration used in the studies (lpM ) is relatively low (Gosule and Schellman, 1976; Widom and Baldwin, 1980). However, at a dilute DNA concentration, DNA condensation can occur in the presence of high spermine concentration (Gosule and Schellman, 1976; Widom and Baldwin, 1980). The counterion condensation theory predicted that the DNA is condensed when about 90% of the phosphate charge is neutralized by polyamines in aqueous solutions (Manning, 1978; Record et al., 1978; Wilson and Bloomfield, 1979; Thomas and Bloomfield, 1983; Bloomfield, 1991). Experimental studies also confirmed this prediction (Gosule and Schellman, 1976,1978; Wilson and Bloomfield, 1979). Based on the above theory, it is possible that the inhibition of crosslink formation may be due to the DNA condensation induced by spermine. This conclusion also explains the high amount of spermine required to inhibit the crosslink formation. Another possible explanation for the inhibition data could be that spermine induces subtle DNA conformation changes, which, in turn, could lead to the formation of DNA duplex structures that are more difficult to crosslink. If the Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 120 spermine-induced DNA duplex conformation change is the cause of crosslink inhibition, the different amounts of spermine required to inhibit each crosslink formation at different crosslinking sites should have been observed. However, the results show no significant difference in the amount of spermine required for 50% inhibition of each crosslink formation. Hence, it is reasonable to believe that a conformational effect is unlikely. The inhibition data provide additional evidence that mechlorethamine C-C crosslink possibly forms in the minor groove and the cytosine bases may become extrahelical in the minor groove of the duplex. Therefore, spermine, which may prefer to bind to a GC-rich major groove or phosphate backbone, cannot push these extrahelical bases located in the minor groove back into the helix. Hence, spermine cannot shift the equilibrium from extrahelical bases to intrahelical bases. The design of molecules, which can specifically bind to the GC-rich minor groove of d[GCC]n*d[GCC]n repeat, may be good candidates in shifting such equilibrium, and may also have potential as Fragile X therapeutic agents. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 121 CHAPTER VI Effects of Cytosine Methylation on Structures of DNA Duplexes Containing a d[GCC]2»d[GCC]2 Fragment: Detection by a Mechlorethamine Crosslinking Reaction 6.1 Introduction As described in Chapter I, Fragile X syndrome is associated with an expansion of d[CGG]n *d[CCG]n triplet repeats within the Fragile X Mental Retardation 1 (FMR1) gene. In the disease state, all the cytosine bases of the d[CG]»d[CG] dinucleotide steps within the d[CGG]n»d[CCG]n repeat region are methylated. Intrastrand hairpins formed by single-stranded d[CCG]n sequence also contain d[CG]*d[CG] sequences which can undergo methylation at the cytosine bases. In such hairpins, it is also possible that the other cytosine bases also become the targets for methylation. Since the cytosine methylation is involved in the development of Fragile X syndrome, it is important to investigate the structures of methylated Fragile X sequence. It has been demonstrated in Chapter IV that duplex I (Figure 6.2) containing two contiguous d[GCC]»d[GCC] helical fragments forms both intrahelical and Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 122 extrahelical cytosine bases w hich can be detected b y m echloretham ine (Figure 6.1). Here, the m echloretham ine crosslinking reaction is used to determ ine structures o f d[C TC TC G X X G X X G C C G T A TC ].d[G A TA C G G C G X X G X X G A G A G ] (duplexes I-VII, Figure 6.2) containing tw o contiguous d[GXX ]»d[G XX] helical fragm ents w here X is cytosine or 5-m ethylcytosine (CM e). The results show that w hen replacing the cytosine w ith 5-m ethylcytosine, the m echloretham ine crosslink form ation at extrahelical bases dim inishes, suggesting that m ethylation at cytosine bases m ay destabilize the eE -m otif D N A conform ation, thus elim inating the extrahelical cytosine bases. c • G B c • G € C • G 1 3 c • S 26 13C • g26 13C • G 2 6 G • c G » C G • c C • G C • G C • G 1 0 C " - M - • C 2 9 10C 0 C 2 9 10C - - M - 'C 2 9 G • c G • c G • c C • G C • G C • G 7C OC3 2 7C - - M - -C 3 2 7* - M - ■ C 3 2 G • C G • c G • C 5' S ' 5' D c • G 13C • G 26 g • c C • G/ ■ 6 • c . A'g * c ’ C 5 - F ig u re 6.1. M echloretham ine-crosslinked species for d[C TC TC G C C G C C G C C G TA - TC ]•d[G A TA C G G C G C C G C C G A G A G ] (D uplex I). The central 9 base pairs o f the duplex are shown, and M — ’ indicates the location o f a m echloretham ine crosslink. A. C10-C29 intrahelical crosslink. B. C7-C32 intrahelical crosslink. C. C10- C29 and C7-C32 double intrahelical crosslink. D. C10-C32 extrahelical crosslink in an extended E -m otif D N A (eE-D N A ) conform ation w ith 5' foldback o f the extrahelical cytosine bases. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 123 I 5 ' -CTCTCGCCGCCGCCGTA TC GAGAGCCGCCGCGGCATAG-5' II 5 ' -C T C T C G C CGC CGCCGTATC GAGAGCCM eGCCMeG CG GCA TA G-5' III 5 ' -C T C T C G C ^C G C ^C G C C G T A T C GAGAGCC GCC G CG G CA TA G -5' IV 5 ' -C T C T C G C ^C G C ^C G C C G T A T C GAGAGCCMeGCC!fcGCGGCATAG-5 ' V 5 ' -C T C T C G CCG CCGCCGTATC GAGAGCMeCGCM eCGCGGCATAG- 5 ' VI 5 ' -C T C T C G C C MeGCCM eGCCGTATC GAGAGCCG CCG C G G C A TA G -5' VII 5 ' -C T C T C G CCM eG CCMeGCCGTATC G A G A G C^CG CMeCG CGGCATAG-5 Figure 6.2. Duplexes I-VII containing two mismatched base pairs at positions 7-32 and 10-32. CM e represents 5-methylcytosine, and mismatch bases are underlines. 6.2 Materials and Methods Chemicals and reagents: Mechlorethamine and T4 polynucleotide kinase were purchased from Sigma. [y-3 2 P]ATP was purchased from ICN. Oligodeoxy- ribonucleotides were synthesized on an Applied Biosystems Model 394 automated synthesizer, deprotected, and purified with a COP cartridge. Other reagents were at least analytical grade. 3 2 P 5 '-end Labeling o f DNA: Column purified synthetic DNA (2.5 pg, 0.5 nanomoles) was 5'-end labeled with [y-3 2 P]ATP (5pi, 4500 Ci/mmol) by incubation Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 124 in buffer (30mM Tris (pH 7.8), 10 mM MgCb, 5mM dithiothreitol) and 30 units of T4 polynucleotide kinase for 1 hour at 37°C. The reaction was stopped by addition of 3M sodium acetate (5.5pL, pH 5.2) and pre-chilled 95% ethanol (150 pL). Unincorporated [y-3 2 P]ATP was removed by precipitation in pre-chilled 95% ethanol (200pL) at -20°C overnight. The labeled DNA was lyophilized and resuspended in distilled water (2pL) and tracking dye (8pL, 80% formamide, ImM EDTA, 0.025% bromophenol blue and xylene cyanol). Purification o f DNA'. The labeled DNA was loaded onto a 20% denaturing polyacrylamide gel (29:1 acrylamide/bisacrylamide, 8M urea, 89mM Tris-borate (pH 8.5) and 2mM EDTA (TBE buffer), 0.4mm thick, 38 x 31 cm, 2500 V, 45 W) until the xylene cyanol marker had migrated 10 cm. The band due to the expected DNA was recovered from the gel using the crush-and-soak procedure. The DNA was then precipitated with ethanol, washed, lyophilized and resuspended in 50 pi of 0.1M NaCl. Annealing o f DNA: The unlabeled complementary strand (2.5 pg) was added to a 50 pi of 0.1M NaCl solution of the purified labeled oligodeoxyribonucleotide, heated to 65°-70°C and then slowly cooled to room temperature. It is noted that the unlabeled strand is in excess, given the less than 100% recovery of the labeled DNA. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 125 Alkylation o f DNA: Following annealing of the strands, a 25 pi of duplex DNA solution was mixed with 0.5 pi of 1M Tris pH 7.5 and 5 pi of 1M NaCl. The mixture was then diluted to 50 pi with purified water and incubated at 37°C with lOOpM mechlorethamine for 2 hours. For each experiment, a fresh solution of lOOmM mechlorethamine was prepared in dimethyl sulfoxide (DMSO), rapidly diluted to lOmM and immediately added to the DNA solution. Following incubation, the reaction was terminated by addition of 3M sodium acetate (5.5pL, pH 5.2), tRNA (5mg/mL, 5 pL) and pre-chilled 95% ethanol (150pL), and precipitated in pre- chilled 95% ethanol (200pL) at -20°C, washed and lyophilized. The DNA was then dissolved in distilled water (2pL) and tracking dye (8pL, 80% formamide, ImM EDTA, 0.025% bromophenol blue and xylene cyanol). Detection and Quantification o f Crosslinked DNA: The samples were loaded onto a 20% denaturing polyacrylamide gel (29:1 acrylamide/bisacrylamide, 8M urea, 89mM Tris-borate (pH 8.5) 2mM EDTA (TBE buffer), 0.4mm thick, 38 x 31 cm, 2500 V, 45 W) until the xylene cyanol marker had migrated for 22 cm. After the gel was exposed to X-ray film, the intensity of each band was quantified using Kodak Digital Science ID software (Kodak Scientific Imaging Systems). Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 126 6.3 Results 6.3.1 A duplex containing two C-C mismatched pairs within a d[GCCGCC]« d[GCCGCC] fragment gives four crosslinked species with mechlorethamine Duplex I (Figure 6.3) containing two C-C mismatched pairs within a d[GCC]2 *d[GCC] 2 repeat was incubated with mechlorethamine and the products of the reaction were separated by electrophoresis on a 20% denaturing polyacrylamide gel (Figure 6.3). Four crosslinked species were formed corresponding to bands Ia-Id (Figure 6.3, lane2). These crosslinked species were previously identified as a C 1 0 -C2 9 intrahelical crosslink (band I/a), a C7 -C3 2 intrahelical crosslink (band I/b), a C 1 0 -C2 9 and C7 -C3 2 double intrahelical crosslink (band I/c), and a C 1 0 -C3 2 extrahelical crosslink (band I/d) (Rojsitthisak et al., 2001). 6.3.2 A duplex containing two C-CM e mismatched pairs within a d[GCCGCC]« d[GCM e CGCM e C] fragment gives five crosslinked species with mechlorethamine. Duplex II (Figure 6.3) containing two C-CM e mismatched pairs within a d[GCCGCC]»d[GCM e CGCM e C] fragment was incubated with mechlorethamine and the products of the reaction were separated by electrophoresis on a 20% denaturing polyacrylamide gel (Figure 6.3). Five crosslinked species were formed corresponding to bands n/a-II/d, Il/r. (Figure 6.3, lane 4). Based on a similar Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Ill mobility pattern to duplex I, four crosslinked bands can be interpreted as a Cio- CMC29 intrahelical crosslink (band Il/a), a C7- CM ®32 intrahelical crosslink (band Il/b), a C 10- CM e29 and C7- CM e32 double intrahelical crosslink (band II/c), and a Cio- CM e32 extrahelical crosslink (band II/d). It is noted that an unexpected band (Il/r) is shown in this duplex, but not in Duplex I. 1 2 3 4 5 6 7 8 9 10 1112 13 14 a a | I « ( l i B iinilB a b lI lH l iiiiB i H IM p b H IM I X > i't m iB i q ? q c c c c c 1 1 1 1 ... c d d d d d d d J M - » s — ► I II ill IV V VI VII Figure 6.3. Autoradiogram of a 20% DP AGE gel showing the products of the incubation of mechlorethamine with duplexes I-VII (Figure 6.2), in lanes 2, 4, 6, 8, 10, 12, and 14, respectively. Lanes 1, 3, 5, 7, 9, 11, and 13 are controls (no mechlorethamine). Bands are identified as X (crosslink), M (monoadduct), and S (unreacted single strands). Each individual band due to a mechlorethamine crosslinked duplex is labeled as a, b, c, d, p, q, and r. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 128 6.3.3 A duplex containing two CM e -C mismatched pairs within a d[GCM e C GCM e C]»d[GCCGCC] fragment gives five crosslinked species with mechlore- th amine. Duplex III (Figure 6.3) containing two CM e -C mismatched pairs within a d[GCM e CGCM e C]®d[GCCGCC] fragment was incubated with mechlorethamine and the products of the reaction were separated by electrophoresis on a 2 0 % denaturing polyacrylamide gel (Figure 6.3). Five crosslinked species were formed corresponding to bands M/a-III/d and ITI/q (Figure 6.3, lane 6 ). Based on a similar mobility pattern to duplex I, four crosslinked bands can be interpreted as a CM eio-C2 9 intrahelical crosslink (band Ill/a), a N 7-C 32 intrahelical crosslink (band Ill/b), a CM eio-C 2 9 and CM V C 3 2 double intrahelical crosslink (band III/c), and a CM eio-C3 2 extrahelical crosslink (band Ill/d). It is noted that an unexpected band (Ill/q) appears in this duplex, but not in Duplex I. 6.3.4 A duplex containing two CM e -CM e mismatched pairs within a d[GCM e C GCM e C]«d[GCM c CGCM e C] fragment gives six crosslinked species with mechlo rethamine. Duplex IV (Figure 6.3) containing two c M e -CM e mismatched pairs within a d[GCM e CGCM eC].d[GCM e CGCM e C] fragment was incubated with mechlorethamine and the products of the reaction were separated by electrophoresis on a 2 0 % Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 129 denaturing polyacrylamide gel (Figure 6.3). Six crosslinked species were formed corresponding to bands IV/a-IV/d, IV/r and IV/q (Figure 6.3, lane 8 ). Based on a similar mobility pattern to duplex I, Four crosslinked bands can be interpreted as a C Meio- C Me29 intrahelical crosslink (band IV/a), a C MV C Me32 intrahelical crosslink (band IV/b), a C Meio- C Me29 and C MV C Me32 double intrahelical crosslink (band IV/c), and a C Meio- C Me32 extrahelical crosslink (band IV/d). Two additional bands which are not observed in duplex I have similar mobilities as bands Il/r and Ill/q (lanes 2 and 4, Figure 6.3). Therefore, the bands are labeled as IV/r and IV/q. 6.3.5 A duplex containing two C-C mismatched pairs within a d[GCCGCC]» [GCCM e GCCM e ] fragment gives five crosslinked species with mechlorethamine. Duplex V (Figure 6.3) containing two C-C mismatched pairs within a d[GCCGCC]»d[GCCM e GCCM e ] fragment was incubated with mechlorethamine and the products of the reaction were separated by electrophoresis on a 2 0 % denaturing polyacrylamide gel (Figure 6.3). Five crosslinked species were formed corresponding to bands V/a-V/d and V/p (Figure 6.3, lane 10). Based on a similar mobility pattern to duplex I, four crosslinked bands can be interpreted as a C 1 0 -C2 9 intrahelical crosslink (band V/a), a C7-C32 intrahelical crosslink (band V/b), a C10- C29 and C7-C32 double intrahelical crosslink (band V/c), and a C10-C32 extrahelical Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 130 crosslink (band V/d). It is noted that an unexpected band (V/p) appears in this duplex, but not in Duplex I. 6.3.6 A duplex containing two C-C mismatched pairs within a d[GCCM e G CCM e ]*d[GCCGCC] fragment gives four crosslinked species with mechlore thamine. Duplex VI (Figure 6.3) containing two C-C mismatched pairs within d[GCCM eGCCM e ]«d[GCCGCC] fragment was incubated with mechlorethamine and the products of the reaction were separated by electrophoresis on a 2 0 % denaturing polyacrylamide gel (Figure 6.3). Four crosslinked species were formed corresponding to bands Vl/a-VI/d (Figure 6.3, lane 12). Based on a similar mobility pattern to duplex I, these four crosslinked bands can be interpreted as a C 1 0 -C2 9 intrahelical crosslink (band Vl/a), a C7-C32 intrahelical crosslink (band Vl/b), a C10- C29 and C7-C32 double intrahelical crosslink (band VI/c), and a C10-C32 extrahelical crosslink (band Vl/d). Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 131 6.3.7 A duplex containing two C-C mismatched pairs within a d[GCCM e G CCM e ]» d[GCCM e GCCM e ] fragment gives six crosslinked species with mechlore thamine. Duplex VII (Figure 6.3) containing two C-C mismatched pairs within a d[GCCM e GCCM e ]»d[GCCM e GCCM e ] repeat was incubated with mechlorethamine and the products of the reaction were separated by electrophoresis on a 20% denaturing polyacrylamide gel (Figure 6.3). Six crosslinked species were formed corresponding to bands VlI/a-VTI/d, VII/p and Vll/q (Figure 6.3, lane 14). Based on a similar mobility pattern to duplex I, four crosslinked bands can be interpreted as a C10-C29 intrahelical crosslink (band VH/a), a C7-C32 intrahelical crosslink (band VH/b), a C10-C29 and C7-C32 double intrahelical crosslink (band VII/c), and a C10-C32 extrahelical crosslink (band VH/d). Two additional bands which are not observed in duplex I have similar mobilities as bands V/p and ffl/q (lanes 2 and 4, Figure 6.3). Therefore, the bands are labeled as VII/p and Vll/q. 6.3.8 Quantification of the mechlorethamine crosslinked duplexes Quantitative data derived for mechlorethamine crosslinking of duplexes I-VII are shown in Table 6.1. The data are shown as the amount of crosslinked DNA, expressed as a percentage of the total DNA, after two hours reaction time. The total crosslink formation of duplexes I-VII ranges from 36-39%, which is consistent with Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 132 previous reports of this reaction in Chapter IV (Rojsitthisak et al., 2001). For duplex I, the crosslink formation for the intrahelical C-C crosslink (bands I/a, I/b and I/c) and the extrahelical C-C crosslink (bands I/d) are 29% and 9%, respectively. These data are also consistent with previous reports of this reaction (Chapter IV) (Rojsitthisak et a l, 2001). However, for duplex II-VII, crosslink formation for extrahelical crosslink of each duplex is less than 2%. For duplexes II, III, V, and VII, unidentified crosslink bands are found with the amount of 6-11% for each band. Table 6.1. Quantification of mechlorethamine crosslinking duplexes I-VIIa % Crosslink1 3 Intrahelical Extrahelical Unidentified Crosslink Crosslink Crosslink Duplex Band c Band a Bandb Q 0 -C2 9 Band d Band p Band q Band r C 1 0 -C2 9 C7 -C3 2 and C 1 0 -C3 2 C7 -C3 2 I 11 11 7 9 0 0 0 II 11 0 7 2 0 0 6 III 11 11 6 <1 0 7 0 IV 13 12 11 <1 0 0 0 V 9 8 9 <1 11 0 0 VI 13 12 11 <1 0 0 0 VII 8 8 6 <1 10 6 0 a Data quantified from the gel shown in Figure 6.3. b The amount of crosslinked DNA for each species, expressed as a percentage of the total DNA. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 133 6.4 Discussion It has been shown previously that C-C mismatched pairs within duplexes containing two contiguous d[GCC]«d[GCC] helical fragments can adopt both intrahelical and extrahelical positions, and these mismatched pairs can be crosslinked by mechlorethamine. The approximate ratio of intrahelical: extrahelical crosslinks is 4:1 (Rojsitthisak et al., 2001) Here, it is shown that, when replacing the cytosines with 5-methylcytosines within a d[GCC]2 »d[GCC] 2 fragment, the mechlorethamine crosslink formation at extrahelical bases diminishes. It is possible that methylation at the 5-position of the cytosine base destabilizes the eE-DNA conformation, thus lowering the amount of extrahelical cytosine bases. This conclusion may also explain why DNA containing extrahelical cytosine bases has a tendency to be methylated because the methylation probably stabilizes such DNA by turning the eE-DNA conformation into a normal DNA duplex structure. It is apparent that duplex II, III, IV, V, and VII give additional crosslink bands with mechlorethamine. It has been shown that mechlorethamine can crosslink a duplex containing d[GCC]«d[GCC] fragment through the 1,3 guanine bases (Romero et. al., 1999, 2001). Therefore, it is possible that these additional bands are the 1,3 G-G crosslinked duplex. If these bands are due to the crosslinking at 1,3 G-G sites, it is possible that methylation at cytosine bases of d[GCC]»d[GCC] fragments Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 134 induce subtle conformational changes of the duplexes and make 1,3 G-G sites more accessible for mechlorethamine. As mentioned in Chapter V, if extrahelical cytosine bases are important in causing promoter hypermethylation, then molecules designed to influence the equilibrium between the intrahelical and extrahelical bases may also have potential as Fragile X therapeutic agents. Hence, the finding that cytosine methylation can alter the equilibrium between the intrahelical and extrahelical cytosine bases may be of value in designing molecules as Fragile X therapeutic agents. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 135 CHAPTER VII Mechlorethamine Crosslinking Reaction with a Hairpin Formed by a d[GCC]5 Single Strand 7.1 Introduction In previous chapters, the structures of d[CCG]n sequence were studied using a mechlorethamine C-C crosslinking reaction with model duplexes containing C-C mismatched pairs within d[GCC]n«d[GCC]n repeats. These duplexes are similar to a hairpin stem formed by d[CCG]n. The results demonstrated that mechlorethamine is able to crosslink such duplexes, leading to an identification of an extended E-motif DNA (eE-DNA) conformation. However, in order to study directly the secondary structures formed by a single-stranded d[CCG]n containing C-C mismatched pairs using mechlorethamine as a probe, it is important to demonstrate that mechlorethamine is capable of crosslinking such structures. This chapter shows that mechlorethamine can crosslink an intramolecular hairpin formed by a single stranded d[GCCGCCGCCGCCGCC] (d[GCC]5 ) sequence. This suggests that mechlorethamine crosslinking reaction may potentially be developed to probe directly the secondary structures formed by d[CCG]n sequence. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 136 7.2 Materials and Methods Chemicals and reagents: Mechlorethamine and T4 polynucleotide kinase were purchased from Sigma. [y-3 2 P]ATP was purchased from ICN. Oligodeoxyribo- nucleotides were synthesized on an Applied Biosystems Model 394 automated synthesizer, deprotected, and purified with a COP cartridge. Other reagents were at least analytical grade. 3 2 P 5 '-end Labeling o f DNA: Column purified synthetic DNA (2.5 pg, 0.5 nanomoles) was 5'-end labeled with [y-3 2 P]ATP (5pi, 4500 Ci/mmol) by incubation in buffer (30mM Tris (pH 7.8), 10 mM MgCl2 , 5mM dithiothreitol) and 30 units of T4 polynucleotide kinase for 1 hour at 37°C. The reaction was stopped by addition of 3M sodium acetate (5.5pL, pH 5.2) and pre-chilled 95% ethanol (150 pL).: Unincorporated [y-3 2 P]ATP was removed by precipitation in pre-chilled 95% ethanol (200pL) at -20°C overnight. The labeled DNA was lyophilized and resuspended in distilled water (2pL) and tracking dye (8pL, 80% formamide, ImM EDTA, 0.025% bromophenol blue and xylene cyanol). Purification o f DNA: The labeled DNA was loaded onto a 20% denaturing polyacrylamide gel (29:1 acrylamide/bisacrylamide, 8M urea, 89mM Tris-borate (pH 8.5) and 2mM EDTA (TBE buffer), 0.4mm thick, 38 x 31 cm, 2500 V, 45 W) until Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 137 the xylene cyanol marker had migrated for 10 cm. The band due to the expected DNA was recovered from the gel using the crush-and-soak procedure. The DNA was then precipitated with ethanol, washed, lyophilized and resuspended in 50 pi of 0.1M NaCl. DNA Hairpin Formation: The purified labeled oligodeoxyribonucleotide was heated to 65°-70°C and then slowly cooled to room temperature overnight. Alkylation o f DNA: A 25 p i of hairpin DNA solution was mixed with 0.5 pi of 1M Tris pH 7.5 and 5 pi of 1M NaCl. The mixture was then diluted to 50 pi with purified water and incubated at 37°C with lOOpM mechlorethamine for 2 hours. For each experiment, a fresh solution of lOOmM mechlorethamine was prepared in dimethyl sulfoxide (DMSO), rapidly diluted to lOmM and immediately added to the DNA solution. Following incubation, the reaction was terminated by addition of 3M sodium acetate (5.5pL, pH 5.2), tRNA (5mg/mL, 5 pL) and pre-chilled 95% ethanol (150pL), and precipitated in pre-chilled 95% ethanol (200pL) at -20°C, washed and lyophilized. The DNA was then dissolved in distilled water (2pL) and tracking dye (8pL, 80% formamide, ImM EDTA, 0.025% bromophenol blue and xylene cyanol). Detection Crosslinked DNA: The samples were loaded onto a 20% denaturing polyacrylamide gel (29:1 acrylamide/bisacrylamide, 8M urea, 89mM Tris-borate (pH Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 138 8.5) and 2mM EDTA (TBE buffer), 0.4mm thick, 38 x 31 cm, 2500 V, 45 W) until the xylene cyanol marker had migrated for 28 cm. Determination o f the Crosslinking Site'. The bands due to the crosslinked DNA were recovered from the gel using the crush-and-soak procedure. The DNA was then precipitated with ethanol, washed, lyophilized, and resuspended in 10% aqueous piperidine in a total volume of lOOpL. To ensure complete cleavage of all alkylated bases, the crosslinked samples were heated for 1 hour at 90°C. For the control Maxam-Gilbert G reaction, the DNA was cleaved using 10% aqueous piperidine, in a total volume of lOOpL, for 20 minutes at 90°C. Different incubation times for the control and mechlorethamine-treated DNA were required because 20 minutes incubation of the crosslinked DNA resulted in insufficient cleavage at the crosslinked sites. All samples were lyophilized overnight, resuspended in 2 pL distilled water and 8pL tracking dye (80% formamide, ImM EDTA, 0.025% bromophenol blue and xylene cyanol), heated at 90°C for 2 min, chilled in an ice bath, and loaded onto a 20% denaturing polyacrylamide gel (29:1 acrylamide/bisacrylamide, 8M urea, 89mM Tris-borate (pH 8.5), 2mM EDTA (TBE buffer), 0.4mm thick, 38 x 31 cm, 2500 V, 45 W) until the xylene cyanol marker had migrated for 10 cm. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 139 7.3 Results 7.3.1 A single-stranded d[GCC]s undergoes the mechlorethamine crosslinking reaction A single-stranded d[GCCGCCGCCGCCGCC] (d[GCC]5 ) was 3 2 P 5'-end labeled and then purified by electrophoresis on a 20% denaturing polyacrylamide gel. The purified DNA was resuspended in 0.1M NaCl and incubated at 65-70°C for 2 minutes and then gradually cooled to room temperature overnight to allow the DNA to form a hairpin. The hairpin was reacted with mechlorethamine and the products of the reaction were separated by electrophoresis on a 20% polyacrylamide denaturing gel. It is shown that there is an additional band ocurred, migrating slightly faster than unreacted DNA. (Figure 7.1, lane 2). 7.4. Discussion It is apparent from the electrophoresis gel (Figure 7.1) that there is an extra band moving faster than the single-stranded d[GCC]5 . It was concluded in Chapter III that the crosslinked species with a compact and stable structure move relatively faster than a loose and unstable structure. Based on the above conclusion, this result suggests that d[GCC]s forms a hairpin structure which is eventually crosslinked by mechlorethamine, giving a tight DNA structure compared to an unreacted single Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 140 stranded DNA. The result leads to the conclusion that the faster band is a crosslinked hairpin DNA. A B s X 1 2 1 2 m . S -► X - > Figure 7.1. Autoradiograms of a 20% DP AGE gel with different exposure time (A and B), showing the mechlorethamine crosslinking of a hairpin formed by a single stranded d[GCC]5 . Lane 1 is a control, showing the hairpin incubated with 0 pM mechlorethamine. Lane 2 shows the hairpin incubated with 100 pM mechlorethamine. Bands due to a single-stranded DNA and a crosslinked hairpin are identified as S and X, respectively. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 141 Two possible structures that can be formed by a d[GCC] 5 single strand are shown in Figure 7.2. Each of them contains two cytosine-cytosine mismatched pairs. If both conformations do exist and each C-C pair is crosslinkable, at least four crosslink bands should be observed. However, the electrophoresis gel shows only one band below the unreacted single-stranded DNA band. This may result from the formation of only one crosslinked species, suggesting that one conformation was formed, and only one C-C mismatched pair of this conformation was crosslinked. Another possible explanation for this result is that more than one crosslinked species was formed, but the electrophoresis condition used here was unable to separate these crosslinks. A B c - C \ / \ 6 c C C \ / V / C • G c • G C o C G • C 6 • —^ C o C C • G c • G C o C G • C G • C C 5 ' 5 ' Figure 7.2. Schematic representation of two possible conformers of d[GCC]s hairpin, showing Watson-Crick pairs (•) and C-C mismatch pairs (o). Hairpin alignment containing A. a d[CG]»d[CG] step and B. a d[GC]»d[GC] step. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 142 There was an attempt to isolate the crosslink band, and to identify the possible crosslinking sites of the DNA using chemical probing and DNA sequencing techniques as described in Chapter IV. However, the experiments failed to identify the crosslinking sites. This was due to an extremely low yield of the crosslinked species, giving an insufficient signal on the sequencing gel for autoradiography. This chapter was able to show that mechlorethamine can crosslink the hairpin formed by d[GCC]5 . However, other separation methods are required for better separation of crosslinked species. Such methods should also be capable of collecting large amounts of DNA. In addition to seeking for other separation methods, further techniques for identifying the structures of crosslinked DNA such as N.M.R. are needed. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 143 CHAPTER VIII High Performance Liquid Chromatography Determination of Mechlorethamine Crosslinking with DNA Duplexes Containing a C-C Mismatched Pair 8.1 Introduction Polyacrylamide gel electrophoresis (PAGE) techniques have been widely used for the isolation and analysis of synthetic oligonucleotides, DNA restriction fragment, polymerase chain reaction (PCR) products, and plasmid. These methods have recently been developed for the detection and separation of interstrand crosslinked DNA and non-crosslinked DNA, including mechlorethamine crosslinked DNA (Hartley et al., 1993; Romero et al., 1999). The methods provide evidence of a novel cytosine-cytosine crosslinking site within the DNA duplex (Romero et al., 1999). Although it is believed that the reaction occurs in the DNA minor groove through N3 atom of cytosine bases (Figure 8.1), the detailed structure of the mechlorethamine C-C crosslinked DNA has not yet been determined (Romero et al., 1999). The reaction has been shown to be useful for probing conformations of DNA containing C-C mismatched pairs (Rojsitthisak et al., 2001). Therefore, it would be Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 144 of benefit to characterize the mechlorethamine C-C crosslinked DNA for the development of this probing reaction. Cl c h 3 I / \ / N \ / \ (a) Cl c h3 5, X I Y C/ V / N v \ c Y X 5 (b) CH. NH. NH, Sugar Sugar Figure 8.1. Mechlorethamine (a) and representation of DNA interstrand crosslinks induced by mechlorethamine at (b) a C-C mismatched pair. Figure (c) shows the probable connectivities of the mechlorethamine crosslink through the cytosine N3 atoms of a C-C mismatched pair. Nuclear magnetic resonance (N.M.R) and X-ray crystallographic techniques have been used to elucidate the detailed structure of DNA. However, the techniques require a highly purified crosslinked DNA and a large amount of sample. Although the PAGE analysis has the advantage of yielding relatively pure DNA and requiring an inexpensive apparatus, the techniques are time consuming, labor intensive, and yield poor recovery. One potential technique is a high performance liquid chromatography (HPLC), which facilitates collecting large quantities of highly purified sample. This will enable a sophisticated N.M.R. and X-ray crystallographic determination of mechlorethamine C-C crosslinked DNA. Here, the HPLC method is Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 145 developed to separate the mechlorethamine crosslinked DNA from non-crosslinked DNA (Figure 8.2). The results show that, using a C4 column and eluting with 5-25% acetonitrile (ACN) in 0.1M triethylammonium acetate (TEAA) and O.lmM ethylenediaminetetraacetic acid (EDTA) over 120 minutes, a HPLC method under these conditions is capable of isolating the mechlorethamine crosslinked DNA from the unreacted DNA. J 5 ' - CTCTCGCCGCCGCCGTATC GAGAGCGGCCGCGGCATAG - 5 ' JJ 5 ' - ATTATATATCTTAATAATA TAATATATACAATTATTAT - 5 ' Figure 8.2. Duplexes containing a C-C mismatched pair. 8.2 Materials and Methods Chemicals: Mechlorethamine (bis(2-chloroethyl)methylamine, nitrogen mustard) was purchased from Sigma. All synthetic oligodeoxyribonucleotides were synthesized on an Applied Biosystems Model 394 automated synthesizer, deprotected, and purified with a COP cartridge at the USC Norris Cancer Center at the University of Southern California. All other reagents were at least analytical grade. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 146 Preparation o f Single-Stranded DNA: Oligodeoxyribonucleotide (25 pM) was prepared in 0.1 M NaCl and 0.05 M Tris (pH 7.5). 100 pi of DNA solution (25 pM) was added with 1 pi of dimethyl sulfoxide (DMSO). The mixture was mixed and 50-pl of the mixture was analyzed using reverse-phase HPLC. Preparation o f Mechlorethamine Crosslinked DNA: 25 pM of a DNA duplex was prepared by mixing an equal amount of complementary oligodeoxyribonucleotides in 0.1 NaCl and 0.05 M Tris (pH 7.5). The solution was heated at 90°C for 2 min and then slowly cooled to room temperature. Following annealing of the strands, 100 pi of duplex DNA solution (25pM) was incubated with 1 pi of mechlorethamine solution (2000 pM) for 2 hours at 25°C. For each experiment, a fresh solution of 2000 pM mechlorethamine was prepared in dimethyl sulfoxide (DMSO) and immediately added to the DNA solution. For control, mechlorethamine solution was replaced with 1 pi of DMSO. Following incubation, 50-pl of the reaction mixture was immediately analyzed using reverse-phase HPLC. HPLC Conditions'. HPLC was performed using a Shimadzu system consisting of two LC-6A pumps, a SPD-6A detector (260nm), a SCL-6A controller and a SIL- 6A auto-injector. The Peak Simple Chromatography Data System (SRI Model 203) was used for data analysis. The system was fitted a reverse-phase Keystone Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 147 Scientific Biobasic-4 column (10 x 250 mm, silica based 5 (am C4 matrix of 300 A pore size). The column temperature was maintained at 65°C using a temperature controller (Cera® Column Heater 250). For gradient elution two buffer systems were used: A buffer [0.1M TEAA and 0.1 mM EDTA, pH 7.0] and B buffer [50% v/v ACN in 0.1M TEAA and 0.1 mM EDTA, pH 7.0]. The column was eluted at a flow- rate of 1.0 ml/min with a linear gradient of B buffer from 10 to 50% in 120 min (5- 25% ACN in 0.1M TEAA and 0.1 mM EDTA, pH 7.0). 8.3 Results 8.3.1 Synthesis and purification of mechlorethamine crosslink of DNA duplex I DNA duplex I (Figure 8.2) was reacted with mechlorethamine, and the mixture was analyzed using a reverse-phase HPLC. The chromatogram shows three significant peaks with the retention time of 23.3, 31.7, and 35.1 minutes (Figure 8.3D). For the identification of these peaks, three control experiments were performed. Two controls containing either top or bottom strands of duplex I (no mechlorethamine) were prepared in the identical buffer as crosslinking reaction mixture. The samples were subjected to HPLC analysis. The top- and bottom- stranded DNA of duplex I show the peaks with the retention times of 24.2 and 22.0 minutes respectively (Figure 8.3A and B). The other control containing both top- and bottom-stranded DNA (no mechlorethamine) was prepared and injected into the Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 148 column. The chromatogram shows two peaks with the retention times of 23.6 and 30.0 minutes (Figure 8.3C). 8.3.2 Synthesis and purification of mechlorethamine crosslink of DNA duplex II DNA duplex II (Figure 8.2) was reacted with mechlorethamine, and the mixture was analyzed using a reverse-phase HPLC. The chromatogram shows four significant peaks with the retention time of 36.3, 36.9, 42.0, and 43.0 minutes (Figure 8.4D). For the identification of these peaks, three control experiments were performed. Two controls containing either top or bottom strands of duplex II (no mechlore-thamine) were prepared in the identical buffer as crosslinking reaction mixture. The samples were subjected to HPLC analysis. The top- and bottom- stranded DNA of duplex n show the peaks with the retention times of 36.4 and 35.8 minutes respectively (Figure 8.4A and B). The other control containing both top- and bottom-stranded DNA (no mechlorethamine) was made and injected into the column. The chromatogram shows two peaks with the retention times of 35.0 and 35.6 minutes (Figure 8.4C). Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 149 24.2 B 22.0 A 30.0 I1 23.6 / D 35.1 31.7 Figure 8.3. HPLC of parent and crosslinked DNA of duplex I (Figure 8.2). Gradient, 5-25% acetonitrile in 0.1M TEAA and O.lmM EDTA, pH 7.0, in 120 min; flow rate 1.0 ml/min. Retention times in min, shown in the chromatogram. A. Top-stranded DNA. B. Bottom-stranded DNA. C. Duplex DNA (no mechlorethamine). D. Duplex DNA with mechlorethamine. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 36.4 » — ' <a» * t - w to t 35.8 35.0,1 3 5 .6 1 .1 ■ t L I . . ! 8 t ) 1 1 ,,1 D 36.3., 36.9 42.2 43.0 / Figure 8.4. HPLC of parent and crosslinked DNA of duplex II (Figure 8.2). Gradient, 5-25% acetonitrile in 0.1M TEAA and O.lmM EDTA, pH 7.0, in 120 min; flow rate 1.0 ml/min. Retention times in min, shown in the chromatogram. A. Top- stranded DNA. B. Bottom-stranded DNA. C. Duplex DNA (no mechlorethamine). D. Duplex DNA with mechlorethamine. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 151 8.4 Discussion The retention time on the reverse-phase column is dependent on the hydrophobicity of the solute. For DNA molecules, its hydrophobicity relies on the polarity of bases and the number of phosphate residues. It has been suggested that, for pyrimidine bases, thymine is more hydrophobic than cytosine because thymine contains a methyl group at the 5-position (Ikuta et al., 1984). For purine bases, guanine is less hydrophobic than adenine probably because guanine contains more polar groups (carbonyl and amino groups) compared to cytosine (an amino group) (Ikuta etal., 1984). In this study, the number of phosphate residues of duplex I and II are the same. Therefore, the difference in the retention time among single strands of the duplexes is mainly due to the hydrophobicity of bases. The results show that the retention time of both strands of duplex II (Figure 8.4A and B) is longer than the retention time of both strands of duplex I (Figure 8.3 A and B). This is because both strands of duplex II (containing AT-rich sequences) are more hydrophobic compared to both strands of duplex I (containing GC-rich sequences). The difference in the retention time between top and bottom strands of duplex I (Figure 8.3A and B, 24.2 and 22.0 minutes for top and bottom strands of duplex I, respectively) is due to the difference in the types of bases. The longer retention time of top strand (Figure 8.3 A, 24.2 minutes) compared to bottom strand (Figure 8.3B, 22.0 minutes) of duplex I Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 152 may be caused by methyl groups of thymine bases within the top strand. For duplex II, there is no difference in the types of bases between top and bottom strands, resulting in the similarity in the retention time of both strands (Figure 8.4A and B, 36.3 and 36.9 minutes for top- and bottom strands of duplex II, respectively). It is of note that the retention times of these strands are slightly different. This suggests that there might be other factors, such as the order of bases within the sequences, responsible for the hydrophobicity of DNA molecules in addition to the types of the bases and the number of phosphate residues. The control containing complementary strands of duplex I (no mechlorethamine) gave two peaks at 23.6 and 30.6 minutes (Figure 8.3C). The first peak at 23.3 minutes (Figure 8.3C) is identified as top-stranded DNA of duplex I consistent with the peak at 24.2 minutes observed in the control containing only top- stranded DNA of duplex I (Figure 8.3A). The loss of the bottom-strand peak and the decrease of top-strand peak suggest that the peak at 30.6 minutes (Figure 8.3C) is the duplex formation of complementary strands of duplex I. In the presence of mechlorethamine, duplex I was crosslinked and the chromatogram of the reaction mixture showed three peaks with the retention time of 23.3, 31.7, and 35.1 minutes (Figure 8.3D). Based on the characterization of controls of duplex I, the peaks at 23.3 and 31.7 minutes (Figure 8.3D) can be identified as top-stranded DNA (Figure 8.3A) and non-crosslinked duplex DNA (Figure 8.3C), respectively. The peak that Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 153 eluted at 35.1 minutes (Figure 8.3D), after parent single strands and the duplex, indicates a product with increased hydrophobicity, suggesting that this peak is a mechlorethamine crosslinked duplex. It is of note that the column temperature of 65°C was not able to denature the duplex structure of duplex I (peaks at 30.0 minutes (Figure 8.3C) and at 31.7 minutes (Figure 8.3D)). Denaturation of DNA duplex structures just prior to injection may be needed to eliminate the duplex strcutures. This would give the crosslink peak without interference. The control containing complementary strands of duplex II (no mechlorethamine) gave two peaks at 35.0 and 35.6 minutes (Figure 8.4C). The first peak at 35.0 minutes (Figure 8.4C) is identified as bottom-stranded DNA of duplex II consistent with the peak at 35.8 minutes observed in the control containing only bottom-stranded DNA of duplex II (Figure 8.4B). The second peak at 35.6 minutes (Figure 8.4C) is identified as top-stranded DNA of duplex II consistent with the peak at 36.4 minutes observed in the control sample containing only top-stranded DNA of duplex II (Figure 8.4A). In the presence of mechlorethamine, duplex II was crosslinked and the chromatogram of the reaction mixture showed four peaks with the retention time of 36.3, 36.9, 42.0, and 42.3 minutes (Figure 8.4D). It is of note that duplex II is completely denatured under the column temperature of 65°C. Based on the characterization of controls of duplex II, the peaks at 36.3 and 36.9 minutes (Figure 8.4D) are identified as bottom-stranded DNA (Figure 8.4B) and top-stranded Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 154 DNA (Figure 8.4A), respectively. The peaks at 42.0 and 42.3 minutes (Figure 8.4D), eluted after parent single strands, indicate two products with increased hydrophobicity, suggesting that these peaks are mechlorethamine crosslinked duplexes. It is interesting to note that duplex II containing only one crosslinking site (C-C mismatched pair) can form two crosslinked duplexes. Formations of two crosslinked species of AT-rich duplexes containing only one C-C mismatched pair (no other possible crosslinking sites) have been previously observed in similar duplexes but detected using gel electrophoresis techniques (Romero et al., 1999). In Chapter II, molecular dynamics simulation results suggest that increasing the number of A-T pairs in the flanking region of a C-C mismatched pair can lead to a local opening of the helix near the C-C mismatch (Romero et al., 2001). Therefore, duplex II which contains AT-rich sequences may allow mechlorethamine to crosslink the C- C mismatch through the major groove in addition to the normal crosslinking reaction in minor groove, resulting in two different crosslinked DNA conformations. The identification of crosslink peaks here is based on the relationship between the hydrophobicity and the retention time of DNA. Other methods such as mass spectrometry, nuclear magnetic resonance spectroscopy, and X-ray crystallography are also needed for further identification and structure elucidation. The HPLC condition developed for separation of crosslinked DNA here will allow for the collection of large amounts of highly purified DNA for such techniques. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 155 CHAPTER IX Discussion 9.1 Sum m ary The cytosine-cytosine (C-C) pair is one of the least stable DNA mismatched pairs. The bases of the C-C mismatch are only weakly hydrogen bonded, and previous work has shown that, in certain sequence contexts, they can become unstacked from the core of the helix, and adopt an ‘extrahelical’ location (Gao et al., 1995). Due to the flexibility of the mismatched cytosine bases, DNA containing C-C mismatched pairs has potential to adopt a variety of unusual DNA conformations. One such example is formation of various conformations of intramolecular hairpins within single-stranded d[CCG]n. These hairpins contain C-C mismatched pairs throughout the structures, and have been linked to triplet repeat expansions and sequence methylations of d[CGG]n»d[CCG]n repeats. Since the expansion and hypermethylation of d[CGG]n«d[CCG]n repeats is associated with the development of Fragile X syndrome, the identification of detailed structures of C-C mismatched pairs within the hairpins formed by single-stranded d[CCG]n may contribute to understanding the molecular basis of the disease. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 156 N.M.R spectroscopy has been widely used for structural determinations of DNA. However, for triplet repeat DNA containing C-C mismatched pairs, this approach is fraught with problems stemming from (a) the repetitive nature of the sequence, which makes spectral assignment difficult, and (b) the conformational dynamics of the DNA, resulting from the flexibility of C-C mismatched pairs within the repeats. It is noted that there has been one previous attempt to study the structures of d[CCG]n»d[CCG]n triplet repeats containing C-C mismatched pairs using N.M.R. techniques (Zheng et al., 1996). It is clear from the study that the above difficulties prevented any meaningful conclusions being drawn, other than that a conformational equilibrium was present in the repeat region (Zheng et al., 1996). One potential approach to study the molecular details of the C-C mismatched pairs within the hairpins formed by single-stranded d[CCG]n is to ‘freeze’ specific conformers by chemically crosslinking the hairpin stems. It was shown previously that mechlorethamine could form an interstrand DNA crosslink at a C-C mismatch pair (Romero et al., 1999). Hence, mechlorethamine may be used to probe the structures of single-stranded d[CCG]n containing C-C mismatched pairs using the above approach. The goal of this dissertation is to identify the structures of C-C mismatched pairs within triplet repeat DNA, using the mechlorethamine C-C crosslinking Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 157 reaction as a chemical probing reaction. To accomplish this goal the kinetics of the mechlorethamine crosslinking reaction at a single C-C mismatched pair within DNA were first determined, in order to identify the appropriate conditions for the probing reactions (Chapter II). In addition, the effects of sequences flanking the C-C mismatched pair on the crosslinking reaction were examined in Chapter II. The results show the rapid kinetics of formation of the C-C crosslink and, in particular, its subsequent resistance to degradation, providing confidence that the reaction will be useful to probe the C-C mismatched pairs in DNA triplet repeats. The results also show that the rate of the reaction and the amount of crosslink formation increase with increasing the G-C content of the bases flanking the C-C mismatched pairs. These results, together with molecular dynamics simulations data, lead to the conclusion that the reactivity of the C-C mismatched pairs towards mechlorethamine is dependent on local stability and conformational fluctuation around the C-C mismatched pair. The differences in crosslinking efficiency may be of value in defining the complex DNA conformations containing multiple C-C mismatched pairs in various locations within the structures (for example, their position relative to a hairpin loop). In performing the kinetics studies, the subtle dependence of electrophoretic mobility on local structural stability and crosslink location was observed. This should also be of value in the separation of the crosslinked species. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 158 The kinetics of the mechlorethamine crosslinking reaction at a single C-C mismatched pair demonstrate the possibility of such reaction for probing hairpin conformations formed by single-stranded d[CCG]n- However, since intrastrand hairpins formed by single-stranded d[CCG]n sequence contain multiple C-C mismatched pairs, other parameters need to be investigated. These are (i) the ability of mechlorethamine to crosslink at a C-C mismatched pair in the presence of other C-C mismatched pairs, (ii) the differences in electrophoretic mobility of various crosslinked products (due to crosslinking at different cytosine bases), that allow the separation to occur, and (iii) the correlation between each crosslinked product and its electrophoretic mobility, which may help in identifying the crosslinking sites of structures. These parameters are investigated in Chapter III. It is shown in Chapter III that mechlorethamine can crosslink a C-C mismatch at various positions on duplex DNA and even multiple, isolated and contiguous mismatched cytosine bases, and the crosslinked products were able to separate on denaturing polyacrylamide gels. However, these crosslinks showed unexpected variable mobility on the gels. The source of this mobility is investigated by examining the mobility of the C-C crosslink in various positions within duplex DNA. It is concluded that the mobility of these crosslinked duplexes depends on the stability of the crosslinked duplexes, particularly at the end of the crosslinked duplexes. The crosslinked duplex with a tight and stable end tends to move relatively Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 159 faster on the gel compared to that with a loose and flexible end. The ability of mechlorethamine to crosslink DNA containing multiple C-C mismatch pairs and the differences in the mobility of each crosslinked duplex imply that that mechlorethamine crosslinking reaction can be used to probe the triplet repeat DNA conformations, and the correlation between the stability of crosslinked products and their electrophoretic mobility will be useful in identifying the crosslinking sites in the complex DNA conformations. After obtaining the appropriate crosslinking reaction and suitable separation conditions, mechlorethamine was used to investigate the structures of C-C mismatched pairs, using a series of DNA duplexes containing d[GCC]n«d[GCC]n repeat fragments (Chapter IV). The d[GCC]n«d[GCC]n repeat fragments chosen here resemble the hairpin stems formed by single-stranded d[CCG]n- The results show that DNA duplexes containing d[GCC]n «d[GCC]n repeat fragments can be crosslinked by mechlorethamine at all formal C-C mismatched pairs, and between specific pairs of cytosine bases that are not formally paired in the duplex structures. The former and latter are interpreted as intrahelical and extrahelical cytosine bases. The detection of extrahelical C-C mismatched pairs provides evidence for an extended E-motif DNA (eE-DNA) conformation. This chapter successfully demonstrates that mechlorethamine can be used as a probe for DNA conformations. These interesting findings extend the dissertation to examine designed molecules that can influence the Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 160 equilibrium between the intrahelical and extrahelical bases, as described below. These molecules may be developed as potential drugs for Fragile X syndrome. In the Fragile X genome all the cytosine bases of the d[CG]»d[CG] dinucleotide steps within the d[CGG]®d[CCG] repeat region are methylated. It is of note that d[CCG]n hairpins are effective substrates for cytosine 5-methyltransferases (Chen et al., 1995) and that these enzymes require the target cytosine to be extrahelical (Klimasauskas et al., 1995). It is possible that the extrahelical cytosine bases may cause eE-DNA to act as a methyltransferase ‘sink,’ and cause the hypermethylation of the proximal promoter region of the FMR1 gene. If extrahelical cytosine bases are important in causing promoter hypermethylation, then molecules designed to influence the equilibrium between the intrahelical and extrahelical bases may also have potential as Fragile X therapeutic agents. The finding in Chapter IV that mechlorethamine can crosslink both intrahelical and extrahelical cytosine bases suggests that mechlorethamine C-C crosslinking reaction can be used as a tool to test whether or not the designed molecules can influence the equilibrium between the intrahelical and extrahelical bases. Polyamines such as spermine have various effects on the DNA structures. Therefore, in Chapter V, spermine was chosen to test its effects on equilibrium between the intrahelical and extrahelical bases using the mechlorethamine C-C Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 161 crosslinking reaction as a probing reaction. The duplex to be used contains two contiguous d[GCC]»d[GCC] helical fragments, which were previously shown to have both intrahelical and extrahelical cytosine bases that could be crosslinked by mechlorethamine. The results show that spermine inhibited both crosslink formations at intrahelical and extrahelical cytosine bases, suggesting the lack of its influence on the equilibrium between the intrahelical and extrahelical bases. The molecules that can bind selectively to certain regions of d[GCC]n*d[GCC]n helical fragments may be needed to exhibit the influence on the equilibrium between the intrahelical and extrahelical bases. As mentioned above, in the Fragile X genome all the cytosine bases of the d[CG]*d[CG] dinucleotide steps within the d[CGG]«d[CCG] repeat region are methylated. Intrastrand hairpins formed by single-stranded d[CCG]n sequences also contain d[CG]»d[CG] sequences which can undergo methylation at the cytosine bases. In such hairpins, it is possible that the other cytosine bases also become the targets for methylation. Since the cytosine methylation is related to the development of Fragile X syndrome, it is important to investigate the structures of methylated Fragile X sequence. It was demonstrated in Chapter IV that the duplex with two contiguous d[GCC]»d[GCC] helical fragments adopts conformations containing intrahelical and extrahelical cytosine bases which can be crosslinked by mechlorethamine. Hence, the mechlorethamine C-C crosslinking reaction is used to Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 162 determine the structures of duplexes containing two contiguous d[GXX]*»d[GXX] helical fragments where X is cytosine or 5-methylcytosine. The results in Chapter VI show that when replacing cytosine with 5-methylcytosine, the mechlorethamine crosslink formation at extrahelical bases diminishes. This suggests that methylation at the 5-position of cytosine base may destabilize the eE-DNA conformation, thus lowering the amount of extrahelical cytosine bases. This conclusion may also explain that DNA containing extrahelical cytosine bases has a tendency to be methylated because the methylation probably stabilize such DNA DNA by turing eE-DNA conformation into stable normal DNA duplex structure. The finding that cytosine methylation can eliminate extrahelical cytosines may be of value in designing molecules as Fragile X therapeutic agents. In Chapter IV, structures formed by single-stranded d[CCG]n sequence were indirectly studied by performing a mechlorethamine C-C crosslinking reaction on model duplexes containing d[GCC]n«d[GCC]n repeats. Duplexes were used, owing to the fact that their structures resemble the stem of a hairpin. However, in order to study directly the hairpin structures formed by a single-stranded d[CCG]n sequence using mechlorethamine as a probe, it is necessary to prove that mechlorethamine can crosslink hairpin structures. Therefore, in Chapter VII, the single-stranded d[GCCGCCGCCGCCGCC] (d[GCC]5 ) is used to test whether or not mechlorethamine can crosslink hairpin structures. The results demonstrate that Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 163 mechlorethamine is able to crosslink an intramolecular hairpin formed by a single stranded d[GCC] 5 sequence, suggesting that mechlorethamine crosslinking reaction can be potentially developed to probe directly the secondary structures formed by d[CCG]n sequence. Crosslinking at a C-C mismatched pair within the DNA duplex by mechlorethamine has been identified using the polyacrylamide gel electrophoresis techniques. Although it has been suggested that the mechlorethamine C-C crosslinking reaction occurs in the DNA minor groove through cytosine N3, the detailed structure of the crosslinked duplex has not yet been determined. Nuclear magnetic resonance (N.M.R.) spectroscopy has been widely used to elucidate DNA structures. However, the technique requires a highly purified crosslinked DNA and large amounts of the sample, which may be difficult to obtain by a gel electrophoresis method. One potential technique is a high performance liquid chromatography (HPLC), which can both purify crosslinked DNA and facilitate collecting large amounts of the sample. In Chapter VIII, an HPLC method is developed to separate the mechlorethamine crosslinked DNA from non-crosslinked DNA. The separation is accomplished using a reverse-phase C-4 column, eluting with 5-25% ACN in 0.1M TEAA and O.lnM EDTA over 120 minutes. This condition may also be developed further for the purification of crosslinked products of secondary conformations formed by single-stranded d[CCG]n- Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 164 9.2 Biological Significance of eE-DNA Conformation This research demonstrates the structural flexibility of C-C mismatched pairs within d[GCC]n»d[GCC]n triplet repeats, resembling the hairpin stems of single stranded d[CCG]n associated with Fragile X syndrome. The C-C mismatched pairs within d[GCC]n »d[GCC]n repeats show their ability to locate at both intrahelical and extrahelical positions. The finding of extrahelical cytosine bases provides evidence for an extended E-motif DNA (eE-DNA) conformation. This stable conformation appears in even a small number of triplet repeats, and tends to increase its amounts in longer repeats. It has been shown that hairpins formed by d[CCG]n repeats are inefficiently repaired, possibly because their structures cannot be recognized by repair enzymes and/or they are poor substrates for repair enzymes (Moore et al., 1996). Hence, unrepaired hairpins may adopt the stable eE-DNA conformation, thereby escaping DNA repair. During replication, if hairpins with the eE-DNA conformation continue to form, and to escape DNA repair, the amount of hairpins will increase. The formation of these hairpins may promote expansions of triplet repeats possibly by DNA slippage mechanism, flap mechanism, or DNA polymerase stalling mechanism. Once the repeats are expanded, the sequence mythylation occurs, causing the development of Fragile X syndrome. It is suggested that d[CCG]n hairpins are effective substrates for cytosine 5-methyltransferases (Chen et al., 1995) and that these enzymes require the target cytosine to be extrahelical (Klimasauskas et al., 1995). Hence, it is possible that the expanded repeats contain multiple hairpins Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 165 with the eE-DNA conformation, making cytosine easily to be methylated. The finding that the eE-DNA conformation can form within d[GCC]n ®d[GCC]n repeats will help in understanding the expansions and hypermethylation of Fragile X triplet repeat DNA. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 166 REFERENCES Aboul-ela F., Koh D., and Tinoco Jr. I. (1985) Base-base mismatches. Thermo dynamics of double helix formation for dCAaXAsG + dCTsYTsG (X,Y = A,C,G,T). Nucleic Acids Res. 13:4811-4824 Ashley C.T. and Warren S.T. (1995) Trinucleotide repeat expansion and human disease. Annu. Rev. Genet. 29:703-728 Ashley C.T., Sutcliffe J.S., Kunst C.B., Leiner H.A., Eichler E.E., Nelson D.L., and Warren S.T. 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Creator Rojsitthisak, Pornchai (author)

Core Title DNA structures associated with the Fragile X triplet repeat sequences

School Graduate School

Degree Doctor of Philosophy

Degree Program Pharmaceutical Sciences

Publisher University of Southern California (original), University of Southern California. Libraries (digital)

Tag biology, molecular,chemistry, biochemistry,OAI-PMH Harvest

Language English

Contributor Digitized by ProQuest (provenance)

Advisor Haworth, Ian S. (committee chair), Bolger, Michael B. (committee member), Hamm-Alvarez, Sarah (committee member), Johnson, Deborah L. (committee member), Shen, Wei-Chiang (committee member)

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c16-544433

Unique identifier UC11334716

Identifier 3094372.pdf (filename),usctheses-c16-544433 (legacy record id)

Legacy Identifier 3094372.pdf

Dmrecord 544433

Document Type Dissertation

Rights Rojsitthisak, Pornchai

Type texts

Source University of Southern California (contributing entity), University of Southern California Dissertations and Theses (collection)

Access Conditions The author retains rights to his/her dissertation, thesis or other graduate work according to U.S. copyright law. Electronic access is being provided by the USC Libraries in agreement with the au...

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