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Different alleles of fission yeast mcm4 uncover different roles in replication

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Different alleles of fission yeast mcm4 uncover different roles in replication

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Content Different alleles of fission yeast mcm4 uncover different roles in replication By Nimna Samarawickrema Ranatunga A Dissertation Presented to the FACULTY OF THE USC GRADUATE SCHOOL UNIVERSITY OF SOUTHERN CALIFORNIA In Partial Fulfillment of the Requirements for the Degree DOCTOR OF PHILOSOPHY (MOLECULAR BIOLOGY) August 2016 Copyright 2016 Nimna S Ranatunga i Acknowledgments The completion of my PhD wouldn’t have been a possibility without the support and contribution of several individuals. I would like to take this opportunity to thank all of those who were involved in various ways to help me achieve this goal. First of all I would like to thank my advisor Dr. Susan Forsburg. I’m extremely fortunate to have had the opportunity to work under Susan. She has been a fantastic mentor, both scientifically and outside of lab. I’m indebted to her for all the advice and help over the years to overcome a number of hurdles to accomplish my final goal. I would like to thank my committee members Dr. Oscar Aparicio, and Dr. Hanna Reisler for their time, advice and support. Spending long hours in lab over the last several years wouldn’t have been possible if not for such a great group of lab mates; who made working in lab a pleasant experience. Thank you to all the past and present members of the Forsburg lab! JiPing Yuan has been a great lab manager making sure that we were stocked up with all the necessary reagents and resources to smoothly carry out experiments. Most of all I appreciate the help given to me whenever I needed extra hands to finish up experiments. Tara Mastro has been a great friend to me from the start of the program. Tara has helped me in numerous ways both inside and outside of lab through my entire graduate school. I would like to thank Tara, for the technical advice, experimental suggestions and for generously been available to answer question and concerns even after leaving the lab. ii Wilber Escorcia and Kuo-Fang Shen have helped me with using the Delta Vision microscope; and patiently shared their knowledge in imaging. Sarah Sabatinos was a great mentor to me over the years. I would like to thank Sarah for teaching me many techniques, and for helping me troubleshoot a number of technical difficulties as a new graduate student. Sarah was a great collaborator as well. Thank you for allowing me to work with you on the mcm4 project (Chapter 2 of the thesis). Marc Green, Pao-Chen Li, Ruben Petreaca and Rebecca Nugent have offered me helpful technical and scientific suggestions. Anh-Huy Le, Lin Ding, Amanda Jensen, and Yael Freiberg have been great lab mates and it has been a pleasure working with you. Dr. Xiaojiang Chen and Ian Slaymaker , thank you for allowing me to collaborate with you on the mcm4 work (Chapter 4 of my thesis). Finally my friends at MCB and USC, have been a great sources of support over the past several year. Special thanks to Anna Skylar my first year mentor, who ended up being a wonderful friend! Thank you for all the help and advice both scientifically and outside of lab. To all my friends at USC; especially to Vivi Tolani, Radheeka Jayasundera and Shanie Liyanagamage who have helped me during different stages at USC. I would also like to thank WiSE for supporting me with funding through parental leave, child care and travel grants. CCLC-UPC (USC childcare) for taking care of my son to help me work my shifts in lab. Last but not the least, none of this would have been humanly possible if not for the support of my family. My wonderful parents have made many sacrifices to help me become who I’m today. They have always encouraged me in pursuing my education and to realize my goals and dreams. A huge thank you to both of them for standing by my side in every step of this journey and for the encouragement and unwavering support. I truly appreciate everything! iii Thank you to my sister Duneesha for all her support and for listing to me when I needed someone to talk! My in-laws for all the help and encouragement given to me at all times to finish my education. Finally I would like to thank my husband Dilanka (DC) Jayasundera & my son Dinira. I truly appreciate all the sacrifices that Dilanka has made to help me finish my PhD. Thank you for being extremely patient with me over the years, keeping up with my weekend lab schedules and bringing me to lab at all odd hours of the night to finish experiments. Most of all listening to my never ending complaints of problematic experiments! I wouldn’t have been able to accomplish this if not for your support. Finally to my two year old son Dinira, who was always there to cheer me up with a smile at the end of the day! iv Table of Contents Acknowledgments i Table of Content iv List of Tables vi List of Figures vii Abstract xi Chapter 1: Introduction 1 Chapter 2: Replication stress in early S phase generates apparent micronuclei and chromosome rearrangement in fission yeast 29 2.1 Introduction 30 2.2 Results 31 2.3 Discussion 36 2.4 Material and Methods 38 2.5 References 39 2.6 Supplemental Material 42 Chapter 3: Characterization of a novel MMS-sensitive allele of S. pombe mcm4+ 61 3.1 Introduction 62 3.2 Material and Methods 64 3.3 Results 68 3.4 Discussion 78 3.5 References 84 3.6 Supplemental Material 117 Chapter 4: Mini-chromosome maintenance complexes form a filament to remodel DNA structure and topology 124 4.1 Introduction 125 4.2 Material and Methods 126 4.3 Results 128 4.4 Discussion 133 4.5 References 134 4.6 Supplemental Material 136 v Appendix A: Identifying potential Cds1 phosphorylation sites on mcm4+ 148 A.1 Purpose 148 A.2 Material and Methods 149 A.3 Results 151 A.4 Conclusion 154 A.5 References 155 Appendix B: Characterizing other mcm4 mutants 156 B.1 Purpose 156 B.2 Material and Methods 156 B.3 Results 157 B.4 Conclusion 160 B.5 References 160 Appendix C: Yeast Two Hybrid (Y2H) to test if the c terminus of mcm4 was responsible in interacting with mcl1 162 C.1 Purpose 162 C.2 Material and Methods 163 C.3 Results 159 C.4 Conclusion 166 C.5 References 168 Appendix D: Determine strand specificity during replication using APOBEC3G as a mutator 169 D.1 Purpose 169 D.2 Material and Methods 170 D.3 Results 173 D.4 Conclusion 176 D.5 References 178 vi List of Tables Table 2.S1: Strains from Forsburg yeast (FY collection) 53 Table 2.S2: Primers used in qPCR reaction 55 Table 3.1: Sensitivity to MMS of double mutants of genes involved in different aspects of the cell cycle 95 Table 3.2: Strains used for this study 100 Table 4.1:X-ray refinement statistics 128 Table 4.S1: Data collection and refinement statistics 137 Table 4.S2: Complementation data at permissive temperature 32 0 C in S. pombe. The wt and mutant MCM4 expressed in the plasmids is driven under an attenuated nmt promoter that is only induced in the absence of thiamine 145 Table 4.S3: A survey of the filament lengths of WT MCM and α5-linker mutant assembled on 1,000 bp dsDNA, showing significant differences 146 Table A.1: Plasmids constructed and used for this work 151 Table A.2: Complementation results 152 Table A.3: Strains used for this study 152 Table B.1: List of plasmids created for this work and complementation results 159 Table B.2: Stains used for this study 159 Table C.1: Cloning vectors for Y2H 164 Table C.2: Y2H plasmids created for the experiments 165 Table C.3: Y2H interaction results 167 Table D.1: Expected mutation patter based on the strand that was exposed 172 Table D.2: Mutation location on untreated mcm4c106 forward and reverse strains 174 Table D.3: Mutation location on the HU treated mcm4-c106 forward and reverse 175 Table D.4: Strains used in this study 177 vii List of Figures Figure 1.1: Assembly of replisome at the Origin Recognition Complex 2 Figure 1.2: Eukaryotic replisome and activity of polymarases during DNA replication 3 Figure 1.3: Components of replication fork complex 5 Figure 1.4: Different regions on the mcm4 N terminal domain 9 Figure 1.5: The sequence alignment of MCM4 protein in S.cerevisea and S.pombe 10 Figure 2.1: Underreplicated mcm4-degron mutants divide during and after replication stress 32 Figure 2.2: Underreplication followed by division promotes micronuclei and genomic rearrangement in fission yeast 34 Figure 2.3: Transient replication instability causes mutation in surviving mcm4-degron cells 35 Figure 2.4: Divisions occur in the presence of DNA damage and repair signals 36 Figure 2.5: Underreplication promotes micronuclei, DNA damage, and aneuploidy in fission yeast 37 Figure 2.S1: Related to figure 1.Characteristics of mcm4-degron and mcm4-ts mutants during temperature shift and release 44 Figure 2.S2: Related to figure 2.Chromosome mis-segregation produces DNA fragments, bridges and centromere separation 47 Figure 2.S3: Related to figure 3. Evidence for mutation at other loci 49 Figure 2.S4: Related to figure 4. The induction of DNA damage and DNA repair signals during and after replication stress 50 viii Figure 2.S5: Related to figure 5. DNA damage, repair and checkpoint control in other replication mutant backgrounds 52 Figure 2.S6: Related to figure 5. DNA damage, repair and checkpoint control through Chk1, Mus81 53 Figure 3.1: Viability of mcm4 mutants at 36° and under MMS treatment 110 Figure 3.2: mcm4-c106 has a defect in replication at permissive temperature 111 Figure 3.3: Replication dynamics in mcm4-c106 at 36° and MMS 112 Figure 3.4: mcm4 protein levels and Chk1 phosphorylation in response to MMS and 36° 113 Figure 3.5: Accumulation of repair foci in mcm4-c106 in response to MMS and temperature 114 Figure 3.6: rif1∆ rescues the mcm4-c106 MMS phenotype 115 Figure 3.7: mcm4-c106 interactions with alternative replication factor C (RFC) 116 Figure 3.S1:Viability of mcm4 mutants treated with HU and CPT 117 Figure 3.S2: mcm4c-106 chromosome segregation during mitosis at 36° using LacI LacO system 118 Figure 3.S3: Effects of Cig2∆ on mcm4-c106 MM S sensitivity 119 Figure 3.S4: mcm4-c106 interactions with genes involved in the error free and error prone repair pathways 120 Figure 3.S5: mcm4-c106 interactions with genes involved in recombination and other pathways 122 Figure 4.1: Overall features of the MCM filament structure 128 Figure 4.2: ET and TEM imaging of MCM–dsDNA filament 120 129 Figure 4.3: The helix α5 rotation and its interaction with the a-subdomain of a neighboring subunit in the filament structure 129 Figure 4.4: The N-terminal domain structural alignment and the DNA topology change induced by MCM 130 ix Figure 4.5: The strong electro-positive ‘strip’ along the helical filament inner surface for DNA binding 131 Figure 4.S1: Samples of electron density map sections 138 Figure 4.S2:Comparison of filament formation and supercoiling of circular plasmid DNA by WT MCM and the α5-linker mutant 139 Figure 4.S3: The mutations of the positively charged residues on the electro-positive DNA binding strip do not disrupt oligomerisation 140 Figure4.S4: Comparison of DNA conformational changes induced by WT and mutant MCM proteins 141 Figure 4.S5: The structure of two neighboring ssoMCM subunits in the filament 142 Figure 4.S6: Gel filtration (Superose-6) chromatography assay of MCM mutants, with molecular marker elution profile shown in panel-c at the bottom 143 Figure 4.S7: Helicase activity of ssoMCM-F540A mutant at the interface between helix α5 and α-domain 144 Figure 4.S8: Phenotypes of mutants (mcm4-Y751A) and wild type mcm4 (mcm4 + ) plasmids transformed in the mcm4 temperature sensitive and in wild type strains 145 Figure 4.S9: Models illustrating how dsDNA (red) binds inside the wide and narrow filaments 147 Figure A.1a: mcm4-4SA containing the 4 point mutants don’t show HU, MMS or temperature sensitivity 153 Figure A.1.b:Relative viability in 12mM Hydroxyurea treatment for 8 hours 154 Figure A.2: Western blot to look at Hydroxyurea specific phosphorylation 154 Figure B.1: Viability in HU treatment 158 Figure B.2: Sensitivity to MMS and HU 158 x Figure C.1: Hypothesis of mcm4 C terminus interacting with mcl1 162 Figure C.2: Yeast Two Hybrid system 156 162 Figure C.3: Mutants tested in the Yeast Two Hybrid assay 164 Figure C.4: Controls for Y2H growth seen on lowest and medium stringency plates supplemented with 3AT to reduce non specific interactions 168 Figure D.1: Signature of mutation created by APOBEC3G 170 Figure D.2: Schematic of the constructs used for the experiment 172 Figure D.3: Growth of APOBEC3G plasmid transformed to Fwd Wild type and Fwd mcm4-c106 173 xi Abstract In this thesis, I’m using Schizosaccharomyces pombe (fission yeast) as a model system to perform an analysis of Mcm4 protein. In Chapter 1, I describe a collaborative project characterizing mcm4-M68 and mcm4- degron. We report that the mcm4 degron mutant is unable to complete DNA replication in S phase, but despite the under-replication, the cells are able to divide at restrictive temperature and release forming structures similar to micronuclei. This was a novel finding and the first report of a micronucleus structure in a yeast system. In Chapter 2, I characterize a truncation mutant of mcm4 that has a temperature sensitive phenotype which also is sensitivity to Methyl methanesulfonate (MMS) an alkylation damaging agent. This mutant lacks 106 amino acids from its C terminus. We see that this mutant functions differently than the canonical mcm4 temperature sensitive alleles (mcm4 M68 & mcm4ts-dg) for temperature sensitivity. The MMS sensitive phenotype has not been observed in other mcm4 mutants, which makes this mutant novel and shows that the C terminus might have a role in maintaining the replisome structure and help with responding to MMS treatment. Finally, in Chapter 3, I describe a collaborative project in which I performed the analysis in the fission yeast system, of a mutation predicted to disrupt a novel MCM filament and its importance in cell growth and survival. 1 Chapter 1 Introduction: Accurate DNA replication is essential for faithful transmission of genetic information. Errors in DNA replication lead to loss of genetic information, chromosome instability, and abnormal cell division. This may ultimately result in cell death, or unregulated cell growth and the generation of cancers. Normal replication may be disrupted by DNA damage. DNA is constantly exposed to damage from endogenous and exogenous sources, therefore cells rely on DNA damage tolerance and repair mechanisms to prevent and repair this damage. 1.1 Replication Fork Establishment: 1.1.1 DNA replication: DNA replication ensures that accurate and identical genetic information is transmitted to daughter cells. This timing of DNA replication is well regulated to ensure that replication occurs only once during synthesis phase (S-phase) of the cell cycle (reviewed in Forsburg, 2004). Replication begins at discrete sites across the genome called origins (Wu & Nurse, 2009, reviewed in Remus & Diffley, 2009). The first step of origin assembly is called licensing. In licensing, replication origins are bound by the Origin Recognition Complex (ORC) (Figure 1). Additional proteins, Cdc18 and Cdt1, associate with the ORC and then the Mini Chromosome Maintenance (MCM) complex is recruited (Bell & Stillman, 1992, Aparicio et al., 1997, Tanaka 2 et al. 1997,Maiorano et al. 2000, Nishitani and Lygerou 2002,Wu & Nurse, 2009). MCM, Cdc18 and Cdt1 together form the pre-replication complex (pre-RC) (Aparicio et al., 1997, reviewed in Forsburg, 2004, S. Tanaka & Araki, 2013). The assembly of the pre-RC marks the origins as competent for replication. The pre-RC assembles only in late M and early G1 phase where there are low levels of CDK activity (reviewed in Nurse,1994).The presence of high levels of CDK in S, early M phase and G2 phase inhibits the pre-RC formation (reviewed in Nurse, 1994, and Nishitani & Lygerou, 2002). This process also ensures that the pre-RC formation as well as the DNA replication occurs only once in the cell cycle. Figure 1.1 Assembly of replisome at the Origin Recognition Complex (from Susan Forsburg) 1.1.2 Helicase and Polymerase function: Eukaryotes are shown to utilize three different polymerases for normal DNA replication and fork progression, Pol α, Polδ and Pol ε (Nick McElhinny et al., 2008, Kunkel & Burgers, 2008 , Johnson et al., 2015, Miyabe, etl.al., 2011, Miyabe et al., 2015). Pol α is known to have limited processivity and lacks the 3’exonuclease activity for proof reading errors. Work carried out in budding yeast and S. pombe have assigned polδ to perform lagging strand synthesis while polε for leading strand synthesis as shown in Figure 2 (Nick McElhinny, et al., 2008, Kunkel & Burgers, 2008). Recent work from Johnson et al show that pol δ is responsible in generating both 3 strands and that the pol ε proof reading is essential to remove any of the errors that are incorporated by pol δ during the process of replication (Johnson et al.,2015). Figure 1.2: Eukaryotic replisome and activity of polymerases during DNA replication. Adapted from (Leman and Noguchi 2013) 1.1.3: MCM proteins: MCM (Mini Chromosome Maintenance) proteins were initially identified in a screen in budding yeast which identified mutants that were defective in the maintenance of a mini chromosome (Maine et al.,1984, reviewd in Tye, 1999, Sinha, et al., 1986). A number of mutants in different genes were isolated with this phenotype, but the name “MCM” has been applied to the six closely related proteins namely Mcm2-7.These are a subgroup of the large AAA ATPase family and are highly conserved in eukaryotes, with some members also found in Archaea (reviewed Forsburg, 2004 ,reviewed Tye, 1999). Metazoans have additional family members Mcm8 and Mcm9 (Yoshida, 2005, Lutzmann et al., 2005, Gozuacik et al., 2003). All MCM proteins share a distinct ATPase motif known as the “MCM Box” (reviewed in Forsburg, 2004, 4 Bochman & Schwacha, 2009, Das et al., 2014, Tye, 1999) that includes consensus ATPase domains called Walker A and Walker B, and the arginine finger. Mcm 2-Mcm7 form a heterohexamer, and all six of these MCMs are essential for viability (Maine et al., 1984, reviewed Forsburg, 2004,Tye, 1999, Bochman & Schwacha, 2010,Das et al., 2014,Hyrien et al.,2003). The MCM complex is associated with the replication fork throughout replication. In vitro the core MCM complex (MCM4/6/7) is sufficient for helicase activity; (Ishimi 1997). However, in vivo the other MCMs as well as other factors such as Cdc45 and GINS are required (Madine et al., 1995, Zhiying et al., 2005). The helicase activity that was observed in vitro was essential to establish the idea that MCM complex functions as the replicative helicase at the fork. Activation of the MCMs and initiation of DNA synthesis, requires CDK (Cyclin Dependent Kinase) and DDK (Dbf4 Dependent Kinase) (Nougarede et. al., 2000, Masai et al., 2006, Sheu and Stillman 2010, Zou and Stillman 1998). These are SpCdc2/ScCdc28 and SpHsk1/Sc Cdc7 respectively. The phosphorylation by the kinases triggers the loading of the additional factors including GINS (Go, Ichi, Nii, San) and Cdc45 (Gambus et al., 2006, Kanemaki & Labib, 2006, Moyer et al., 2006) which activate the MCMs as an unwinding complex. Cdc45, GINS and MCM (CMG complex) all move with the replication fork (Moyer et al., 2006, Gambus et al., 2006, Evrin et al., 2009, Ilves et al., 2010, Kanemaki & Labib, 2006, Reviewed in Das et al., 2014, Bochman & Schwacha, 2009). Early work (Laskey & Madine, 2003) showed that the number of MCM molecules exceed origins and they broadly associated with unreplicated DNA. Some of the “excess” MCM complexes license dormant origins play an important role in maintaining genomic integrity under conditions of replicative stress (Ibarra et 5 al., 2008). Others are not associated with origins at all, leading to the speculation that they may mark unreplicated chromatin. 1.1.4 Fork protection complex: Additional factors are needed to maintain the proper structure and function of the replisome. The fork protection complex (FPC) is one such structure (Figure 3). FPC is composed of spSwi1 (ScTof1/TIM1), spSwi3 (ScCsm3/TIPIN), and spMrc1 (Sc Mrc1/CLASPIN). As the name suggests, this complex helps to stabilize the replication fork, and also has a role in fork pausing and fork termination (Noguchi et al., 2004, Dalgaard & Klar, 2000). Swi1-Swi3 complex is essential when lagging-strand synthesis is compromised through a mutation in pol δ (Noguchi et al., 2004). The replisome is further coupled by SpMcl1 (ScCtf4), which tethers the CMG to DNA polymerase alpha (Pol α) (Simon et al., 2014, Zhu et al., 2007,Gambus et al., 2009). This is confirmed by structural evidence (Simon et al., 2014). This relationship has been shown biochemically in S. pombe, where Mcl1 was shown to bind to the N- terminus of Polα during S phase and in DNA damage (Williams and McIntosh 2002). SpMcl1 also plays a role in cohesion ensuring proper chromosome segregation (Williams & McIntosh 2002, Williams & McIntosh, 2005), while showing a genetic interaction with DDK (Williams & McIntosh 2002). 6 Figure 1.3: Components of replication fork complex. Adapted from (Gambus et al., 2009) 1.2 Importance of MCMs: 1.2.1 MCM in cancer: Mutations in Mcm4 are associated with different types of cancers. Point mutation F345I mcm4 chaos3 mutation near the Zn-finger motif of MCM4 is associated with mammary carcinoma in mice (Shima et al.,2007a, Shima et al., 2007 b) and mcm4-D573H is associated with T cell lymphoblastic leukemia/lymphoma in mouse models (Bagley et al., 2012). G364R mutation on MCM4 in humans have been shown to be associated with skin cancer (Ishimi and Irie 2015). N-terminally truncated Mcm4 (∆1-50 ) is linked to glucocorticoid deficiency, and atypical Fanconi Anemia in humans (Hughes et al., 2012,Gineau et al., 2012). Studies have shown that MCM proteins are increased in cells from cancerous/malignant tissue compared to normal tissues (Hua et al., 2014,Y Ishimi et al., 2003, Freeman et al., 1999, Tan et al., 2001, Lei et al., 2005). MCMs have not been reported to be abundantly expressed in differentiated somatic 7 cells. Given this specificity, MCMs are predicted to be a useful marker in detection as well as targeting anticancer drug development (Lei et al., 2005). 1.2.2 Mutant alleles of S. pombe mcm4 + : The mcm4 + (cdc21 + ) gene was originally identified in a screen for temperature sensitive cdc mutants that arrest as elongated cells with undivided nuclei (Nasmyth & Nurse, 1981; Coxon et al., 1992). The original allele mcm4-M68 carries a single amino acid substitution (Leu238 to Pro), that could presumably lose its function at a high temperature through a conformational change in the protein structure (Nasmyth and Nurse 1981). Cells undergo a checkpoint- dependent arrest at restrictive temperature with an apparent 2C DNA content and show evidence of DNA damage including DNA double strand breaks (Nasmyth & Nurse, 1981, Coxon et al., 1992, Liang et al., 1997, Bailis et al., 2008). Chromosomes show defective migration in pulsed field gels, indicating breakage and unresolved replication and/or recombination intermediates (Liang & Forsburg, 2001, Sabatinos et al., 2015). Viability is low upon return to permissive temperature, suggesting this damage is irreversible (Liang & Forsburg, 2001, Bailis et al., 2008). Cells carry visible signs of damage including foci containing the single strand DNA binding protein RPA and the recombination protein Rad52 (Bailis et al., 2008, Sabatinos et al., 2015). The phenotype of late S-phase arrest suggests that mcm4-M68 cells are competent for replication initiation and progression at the restrictive temperature, with late replication fork break down. This may occur at specific fragile regions, or be stochastically distributed. A second temperature sensitive allele was constructed by fusing a degron cassette to mcm4-M68 (Kearsey et al., 2000). This enhances protein turn-over leading to a rapid inactivation at restrictive temperature and cells arrest with a 1C DNA content (Lindner etl al., 2001, 2002, 8 Bailis et al., 2008). However, despite evidence of DNA damage including large RPA- and Rad52-containing “megafoci”, these cells continue to divide, indicating that they have evaded the checkpoint (Sabatinos et al., 2015). Previously, we constructed several targeted point mutations in conserved residues of Mcm4. Mutation of conserved residues in the Walker A and Walker B motif abolish Mcm4 activity (Gómez et al.,2002). Mutations in the consensus CDK phosphorylation sites have no obvious phenotype (Gómez et al.,2002). Nitani et al (Nitani et al., 2008) constructed C-terminal truncations of mcm4 and showed that the C-terminus is required to restrain single-strand DNA accumulation. One of these alleles, mcm4-c106, also proved to be temperature sensitive and its characterization will be the subject of Chapter 3. 1.2.3 The Mcm4 protein is a central target of regulation: Mcm4 has been identified as a key substrate for regulation of replication. The N terminus of mcm4 undergoes DDK-dependent phosphorylation both in vivo and in vitro. This has been shown by work done on both fission and budding yeast systems. (Masai et al., 2006, Sheu et al.,2014, Sheu & Stillman, 2006, Sheu & Stillman, 2010). There are putative CDK sites that are present in the N terminus region of the mcm4. Mutational analysis studies carried out on the CDK phosphorylation site 15 and 112 in S.pombe Mcm4 show that despite the mutation that they were able to undergo phosphorylation and had no effect on replication (Gomez et al., 2002). Structure-function analysis of the N terminus of mcm4 has been extensively carried out in budding yeast. As shown in the schematic below (Figure 4) the N terminus region can be 9 divided into two segments; the proximal and the distal, each of which has a distinct function (Sheu & Stillman, 2010,Sheu et al., 2014). Figure 1.4: Different regions on the mcm4 N terminal domain in S.Cerevisiae. (Adapted from Sheu et al., 2010/2014) Sac MSQQSSSPTKEDNNSSSPVVPNPDSVPPQLSSPALFYSSSSSQGDIYGRNNSQNLSQGEG pombe --MSSSQQSGRANELRTPGRANSSSREAVDSSPLFFPASSPGSTRLTTPRTTARTPLASS .**. : . *: :* .*..* . *** :* :**... : ..: . . ... Sac NIRAAIGSSPLNFPSSSQRQNSDVFQSQGRQGRIRSSASASGRSRYHSDLRSDRALPTSS pombe PLLFESSSPGPNIPQSSR----SHLLSQRNDLFLDSSSQRTPRSTRRGDIHSSVQMSTPS : .*. *:*.**: . : ** .: : **:. : ** :.*::*. :.*.* Sac SSLGRNGQNRVHMRRNDIHTSDLSSPRRIVDFDTRSGVNTLDTSSSSAPPSEASEPLRII pombe -----------RRREVDPQRPGVSTP----SSLLFSGSDALTFSQAHPSSEVADDTVRVI : *. * : ..:*:* . ** ::* *.: .... *.:.:*:* Sac WGTNVSIQECTTNFRNFLMSFKYKFRKILDEREEFINNTTDEELYYIKQLNEMRELGTSN 10 pombe WGTNVSIQESIASFRGFLRGFKKKYRPEYRN--ELMPPPDAEQLVYIEALRNMRIMGLEI *********. :.**.** .** *:* : *:: . *:* **: *.:** :* . Sac LNLDARNLLAYKQTEDLYHQLLNYPQEVISIMDQTIKDCMVSLIVDNNLDYDLDEIETKF pombe LNLDVQDLKHYPPTKKLYHQLYSYPQEIIPIMDQTIKDVMLDLLGTNPPEDVLNDIELKI ****.::* * *:.***** .****:*.******** *:.*: * : *::** *: Sac YKVRPYNVGSCKGMRELNPNDIDKLINLKGLVLRSTPVIPDMKVAFFKCNVCDHTMAVEI pombe YKIRPFNLEKCINMRDLNPGDIDKLISIKGLVLRCTPVIPDMKQAFFRCSVCGHCVTVEI **:**:*: .* .**:***.******.:******.******** ***:*.**.* ::*** Sac DRGVIQEPARCERIDCNEPNSMSLIHNRCSFADKQVIKLQETPDFVPDGQTPHSISLCVY pombe DRGRIAEPIKCPREVCGATNAMQLIHNRSEFADKQVIKLQETPDVVPDGQTPHSVSLCVY *** * ** :* * *. .*:*.*****..**************.*********:***** Sac DELVDSCRAGDRIEVTGTFRSIPIRANSRQRVLKSLYKTYVDVVHVKKVSDKRLDVDTST pombe DELVDSARAGDRIEVTGIFRCVPVRLNPRMRTVKSLFKTYVDVVHIKKQDKRRLGTDPST ******.********** **.:*:* *.* *.:***:********:** ..:**..*.** Sac IEQELMQNKVDHNEVEEVRQITDQDLAKIREVAAREDLYSLLARSIAPSIYELEDVKKGI pombe LESDIAEDAALQ--IDEVRKISDEEVEKIQQVSKRDDIYDILSRSLAPSIYEMDDVKKGL :*.:: :: . : ::***:*:*::: **::*: *:*:*.:*:**:******::*****: Sac LLQLFGGTNKTFTKGG--RYRGDINILLCGDPSTSKSQILQYVHKITPRGVYTSGKGSSA pombe LLQLFGGTNKSFHKGASPRYRGDINILMCGDPSTSKSQILKYVHKIAPRGVYTSGKGSSA **********:* **. *********:************:*****:************* Sac VGLTAYITRDVDTKQLVLESGALVLSDGGVCCIDEFDKMSDSTRSVLHEVMEQQTISIAK pombe VGLTAYITRDQDTKQLVLESGALVLSDGGICCIDEFDKMSDATRSILHEVMEQQTVTVAK ********** ******************:***********:***:*********:::** Sac AGIITTLNARSSILASANPIGSRYNPNLPVTENIDLPPPLLSRFDLVYLVLDKVDEKNDR pombe AGIITTLNARTSILASANPIGSKYNPDLPVTKNIDLPPTLLSRFDLVYLILDRVDETLDR **********:***********:***:****:******.**********:**:***. ** Sac ELAKHLTNLYLEDKPEHISQDDVLPVEFLTMYISYAKEHIHPIITEAAKTELVRAYVGMR pombe KLANHIVSMYMEDTPEHATDMEVFSVEFLTSYITYARNNINPVISEEAAKELVNAYVGMR :**:*:..:*:**.*** :: :*:.***** **:**:::*:*:*:* * .***.****** Sac KMGDDSRSDEKRITATTRQLESMIRLAEAHAKMKLKNVVELEDVQEAVRLIRSAIKDYAT pombe KLGEDVRASEKRITATTRQLESMIRLSEAHAKMHLRNVVEVGDVLEAARLIKTAIKDYAT *:*:* *:.*****************:******:*:****: ** **.***::******* Sac DPKTGKIDMNLVQTGKSVIQRKLQEDLSREIMNVLKDQASDSMSFNELIKQINEHSQDRV pombe DPATGKISLDLIYVNERETLVPEDMVKELANLISNLTVGGKTMLVSQLLTRFREQSSTRL ** ****.::*: ..: : . : ...:* ..:*:.::.*:*. *: Sac ESSDIQEALSRLQQEDKVIVLGEGVRRSVRLNNRV- pombe DASDFEACLGALERRGRIKVITSAGHRIVRSIAQTD ::**:: .*. *::..:: *: .. :* ** :. Figure 1.5 The sequence alignment of MCM4 protein in S.cerevisea and S.pombe 11 Above is the MCM4 protein alignment of the S.cerevisea and S.pombe. The region indicated in red is the N terminus. The conserved amino acids are marked with an asterisk (*). It can be observed that there is only a very limited amount of conserved amino acids between the N terminal regions of the two organisms. The proximal segment of the NSD which lies between (amino acids 74-174) has been shown to have a function in the initiation inhibitory activity that is mitigated by DDK through phosphorylation where as the distal portion of mcm4 which lies between the segments (2-145) has been shown to have an important function in the fork progression when cells have depleted nucleotide pools as is the case during hydroxyurea (HU) treatment (Devault et al., 2008; Sheu et al., 2014). Additionally mcm4- Δ2-174 has been shown to bypass DDK function (Sheu and Stillman., 2010). 1. 3: Replication fork impediments: 1.3.1 Replication stress in response to external agents: Replication stress is a general term that describes challenges to efficient DNA synthesis, often imposed by external agents. In fission yeast, there are drugs used to create replication stress and these have distinct effects on S phase progression. Hydroxyurea (HU) inhibits nucleotide synthesis, which depletes nucleotides and causes fork stalling (Thelander & Reichard, 1979,Koç et al., 2004). The normal response to HU treatment leads to arrest early in S phase with limited DNA synthesis from early replication origins (Alcasabas et al., 2001, Santocanale & Diffley 1998, Santocanale 1998, Sogo et al., 2002, Sabatinos et al., 2012). The Cds1 checkpoint is important to preserve the replication fork during fork stalling and also to prevent late origin firing and mitotic entry; in its absence, DNA 12 unwinding and DNA synthesis continue with catastrophic results (Sabatinos et.al., 2012). In the absence of Cds1, replication forks collapse, generating double strand breaks and activating the damage checkpoint (Lindsay et al., 1998, Martinho et al., 1998, Bailis et al., 2008). Camptothecin (CPT) is a topoisomerase type I inhibitor that leads to S-phase specific double strand breaks (Eng et al.,1988, Liu et al., 2000). These breaks are recognized by the DNA damage checkpoint, leading to activation of Chk1, and cell cycle arrest (Wang et al., 1999). MMS is a methylating agent that generates diverse lesions that blocks polymerases mainly by introducing DNA adducts in the form of methyl groups on DNA bases (Wyatt & Pittman, 2006, Lundin et al., 2005,Drabløs et al., 2004). These can block replicative helicases, and polymerases and can cause replication fork stalling (Branzei & Foiani, 2009, Jossen & Bermejo, 2013). There have been reports indicating that MMS is capable of causing DSBs (Wyatt &Pittman 2006);.however, this breakage may actually be an artifact of the procedure used in preparing DNA for Pulse Field Gel Electrophoresis (Lundin et al., 2005). MMS treatment does cause the activation of the intra S phase checkpoint and during S phase the damage is repaired by several mechanisms, including homologous recombination, template switching, and translesion synthesis pathways (Branzei & Foiani, 2009, Branzei & Foiani, 2010). MMS slows the S phase progression of cells, by inhibiting the late origin firing and also by slowing the fork progression (Kumar & Huberman, 2009,Willis & Rhind, 2009). Work carried out in budding yeast has shown that that the slowing of the replication fork rate is independent of the Mec1 and Rad53, (Rad3 and Cds1 in S pombe respectively), where as in S. pombe it appears to be dependent on both Rad3 and Cds1 (Kumar & Huberman 2009). Cells 13 with mutations in the replication fork protection complex (Swi1,Swi3) are sensitive to MMS and HU treatment (Noguchi et al., 2003; Noguchi et al., 2004, Sommariva et al., 2005). 1.4 Replication fork repair and restart: 1.4.1 Checkpoints: The S. pombe cell cycle has distinct cell cycle stages G1, S, G2 and M phase. When cells encounter DNA damage at any of the above mentioned stages of the cell cycle, a checkpoint is activated. This signal ensures that the cell does not proceed with the cell cycle until the damage is repaired (Hartwell & Weinert, 1989, Rhind & Russell, 1998). If a cell lacks a gene that is responsible for activation or control of the damage checkpoint, the cell will proceed through the cell cycle in the presence of the damage which can lead to cell death, more DNA damage, or unregulated cell growth. Environmental factors such as UV, IR and genotoxic drugs can cause replication stress (reviewed Branzei & Foiani, 2008, 2009, 2010, Zeman & Cimprich, 2014). Apart from these external sources of damage, intrinsic conditions such as, repeated DNA sequences, proteins bound to DNA, chromatin compaction, exposed ssDNA, and collisions occurring between RNA polymerases and the replication fork can also induce damage and activate a checkpoint (reviewed in Zeman & Cimprich, 2014,Pomerantz & O’Donnell, 2010). Regardless of the type or the source of damage these generate replication stresses, which in turn lead to genomic instability. Cells have adapted different mechanisms to respond to these damages and stresses, mainly though the activation of the damage checkpoint. There are two pathways that respond to DNA damage in fission yeast, which are controlled by the upstream kinase Rad3 (Sc Mec1) as the master regulator. Downstream of Rad3, there are two kinases involved in different pathways Chk1 and Cds1. Response to DNA damage 14 and DNA breaks is enforced by the kinase Chk1, often called the G2 or damage checkpoint. Cds1 enforces the replication checkpoint including response to replication stress (reviewed Thomas Caspari & Carr, 1999, Rhind & Russell, 1998). Rad3 gets recruited to the sites of damage by the 9-1-1 complex. This complex is composed of Rad9, Hus1, and Rad1. In the presence of damage, the 9-1-1 complex is loaded around DNA (Kaur et.al 2001, Volkmer & Karnitz, 1999) and causes the ATR-mediated phosphorylation and activation of Chk1. Apart from the activation of the Chk1 kinases, 9-1-1 complex has been shown to have a role in DNA repair (T Caspari et al., 2000, reviewed in Helt et al., 2005). Single stranded DNA (ssDNA) bound by the protein RPA (replication protein A) is an important signal indicating DNA damage (Choi et al., 2010, Lee Zou & Elledge, 2003). There is modest accumulation of ssDNA during DNA replication, but in response to fork stress or damage, the MCM helicase and polymerase s may become uncoupled, leading to increased ssDNA and RPA signaling. In humans it has been shown that RPA binding to ssDNA is required for the recruitment of ATR to sites of DNA damage and for ATR-mediated Chk1 activation (Lee Zou & Elledge, 2003, Z. You et al., 2002 ). Cellular replication can be delayed or arrested in two proposed ways with the checkpoint activation. Checkpoint activation can slow bulk replication by inhibiting replication origin firing or it could slow replication fork progression. Both of these mechanisms take place as a result of the S phase DNA damage checkpoint (Willis & Rhind, 2009, Kumar & Huberman, 2004, 2009). 15 1.4.2 DNA Damage Repair: Failure to manage replication stress can lead to DNA damage and activation of repair pathways. Inability to repair these damages in cells can cause chromosome and genomic instability and cell death (Friedberg, 2005,Ceccaldi et al., 2015). Briefly, there are multiple mechanisms of repair. Homologous recombination (HR) and non homologous end joining (NHEJ) are the two main mechanism utilized in the repair of DSB (double stranded breaks). base excision repair (BER), nucleotide excision repair (NER), mismatch repair (MMR), post replication repair (PRR) are other mechanistic pathways. BER is the predominant method of repair for alkylation damages (MMS) (Memisoglu and Samson 2000) and oxidative stress. NER is mainly used to repair helix-distorting damage caused by environmental mutagens such as UV induced breaks (Lindahl and Wood 1999). In fission yeast it has been shown that BER and NER work together to repair MMS induced breaks while post replication repair pathways play a role in this process as well (Kanamitsu & Ikeda, 2011, reviewed in Friedberg, 2005, Memisoglu & Samson, 2000). Error free and error prone are two other post replication repair pathways used in DNA damage repair. These methods are used mainly when there is damage present in the template DNA. (Uchiyama et al., 2015). In fission yeast, Eso1 (polη), Kpa1/DinB (polκ), Rev1, and polζ (a complex of Rev3 and Rev7) have been identified as translesion synthesis (TLS) polymerases (Uchiyama et al., 2015, Prakash et al., 2005). The error prone TLS polymerases normally replicate across a damages section. Template switch (TS), which is error-free; utilizes the 16 undamaged information of the sister duplex to bypass the damage (Branzei & Foiani, 2009, Lazzaro et. al., 2009). 1.5 Conclusion: In the following chapters I describe the work carried out on different alleles of fission yeast mcm4 and its role in DNA replication. In chapter 1 I show the work that was carried out in collaboration with Dr.Sarah Sabatinos a former post doc in our lab. We studied the well established mcm4ts (mcm4-M68) and mcm4ts-degron mutations. We discovered that although mcm4-M68 and mcm4ts-dg both harbor the same point mutation they have very distinct phenotypes in DNA replication. We showed that mcm4-degron evade the damage checkpoint and go through DNA replication in the presence of damage; and produce structures similar to micronuclei (Sabatinos et.al., 2015). In chapter 2, I examined a C terminal truncation of mcm4 that removes 106 amino acids of the C terminus. 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(2015) Replication stress in early S phase generates apparent micronuclei and chromosome rearrangement in fission yeast. Mol Biol Cell. 2015 Oct 1;26(19):3439-50. I contributed towards the preliminary work that was done for the project by performing viability assays, and DAPI staining to access physiological changes in strains under different treatment conditions. For the publication, I contributed by carrying out pulsed field gel electrophoresis (PFGE) (Supplementary Figure 4f) relative viability assays, and flow cytometry for the different experiments which are shown in figure 1 and supplementary figure 1 Volume 26 October 1, 2015 3439 MB oC | ARTICLE Replication stress in early S phase generates apparent micronuclei and chromosome rearrangement in fission yeast ABSTRACT DNA replication stress causes genome mutations, rearrangements, and chromo- some missegregation, which are implicated in cancer. We analyze a fission yeast mutant that is unable to complete S phase due to a defective subunit of the MCM helicase. Despite underreplicated and damaged DNA, these cells evade the G2 damage checkpoint to form ultrafine bridges, fragmented centromeres, and uneven chromosome segregations that resembles micronuclei. These micronuclei retain DNA damage markers and frequently rejoin with the parent nucleus. Surviving cells show an increased rate of mutation and chromosome rearrangement. This first report of micronucleus-like segregation in a yeast replication mutant establishes underreplication as an important factor contributing to checkpoint escape, abnor- mal chromosome segregation, and chromosome instability. INTRODUCTION DNA replication stress is a well-known source of genome instability and results in increased mutations, chromosome rearrangements, and missegregation (reviewed in Naim and Rosselli, 2009; Crasta et al., 2012; Holland and Cleveland, 2012; Hatch et al., 2013). Tem- pering replication stress by adding extra nucleosides (Burrell et al., 2013) or inducing a checkpoint response (Casper et al., 2002) can stabilize slowly replicated regions and diminish the effect on chro- mosome missegregation. Of importance, genome instability is also correlated with carcinogenesis (e.g., Bagley et al., 2012; Burrell et al., 2013; Hirsch et al., 2013), particularly within fragile regions of the genome that are unable to replicate efficiently (e.g., Chan et al., 2009; Lukas et al., 2011; Naim et al., 2013). Thus cellular ability to appropriately manage replication stress prevents malignant trans- formation (Bartkova et al., 2005; Gorgoulis et al., 2005; Halazonetis et al., 2008). MCM4 is an essential subunit of the minichromosome mainte- nance (MCM) helicase that is required for DNA replication (reviewed in Forsburg, 2004; Bochman et al., 2008). Mice with minor mcm4 mutations show evidence of replication stress, including double- strand breaks, micronuclei, and increased formation of mammary tumors (Shima et al., 2007a) or leukemia (Bagley et al., 2012). Disrup- tions in replication correlate with chromosome fragile sites (reviewed in Debatisse et al., 2012), and the murine mcm4 phenotype is con- sistent with a failure to license dormant replication origins (reviewed in Kawabata et al., 2011; McIntosh and Blow, 2012). N-terminal trun- cation of Mcm4 is associated with chromosome breaks and DNA repair defects in an inbred human population (Casey et al., 2012; Gineau et al., 2012; Hughes et al., 2012). MCM overexpression has been correlated with hyperproliferation and carcinogenesis in tu- mors (Ishimi et al., 2003b; Guida et al., 2005; Lau et al., 2010; Majid et al., 2010; Suzuki et al., 2012). Thus changes in this single MCM4 subunit have profound consequences for genome stability. We report a novel genome-instability phenotype in a specific allele of mcm4. In fission yeast cells, most conditional mcm muta- tions at the restrictive temperature show significant DNA accumula - tion, accompanied by activation of the DNA damage checkpoint and robust cell cycle arrest (e.g., Nasmyth and Nurse, 1981; Coxon et al., 1992; Liang and Forsburg, 2001) consistent with replication Monitoring Editor Mark J. Solomon Yale University Received: May 28, 2015 Revised: Jul 24, 2015 Accepted: Jul 24, 2015 This article was published online ahead of print in MBoC in Press (http://www .molbiolcell.org/cgi/doi/10.1091/mbc.E15-05-0318) on August 5, 2015. S.A.S. and S.L.F. designed the study; S.A.S., N.S.R., J.P .Y., and M.D.G. performed the experiments or analyzed the data; and S.A.S. and S.L.F . wrote the manuscript. Address correspondence to: Susan L. Forsburg (forsburg@usc.edu). © 2015 Sabatinos et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is avail- able to the public under an Attribution–Noncommercial–Share Alike 3.0 Unport- ed Creative Commons License (http://creativecommons.org/licenses/by-nc -sa/3.0). “ASCB ® ,” “The American Society for Cell Biology ® ,” and “Molecular Biology of the Cell ® ” are registered trademarks of The American Society for Cell Biology. Abbreviations used: A.U, arbitrary units; BIR, break-induced replication; CFP , cyan fluorescent protein; cut, cell untimely torn; 3D-SIM, 3-dimensional structured illu - mination microscopy; EdU, 5-ethynyl-2′-deoxyuridine; FUdR, 5-fluoro-2′-deoxyu- ridine; GCR, gross chromosomal rearrangements; GFP , green fluorescent protein; HU, hydroxyurea; iso, isochromosome; MCM, minichromosome maintenance; MN, micronucleus; MUG, mitosis with unreplicated genomes; PMG, pombe min- imal medium with glutamate; PN, parent nucleus; RFP, red fluorescent protein; RPA, replication protein A; ssDNA, single-strand DNA; UFB, ultrafine bridge; wt, wild type; YFP , yellow fluorescent protein. Sarah A. Sabatinos a,b , Nimna S. Ranatunga a , Ji-Ping Yuan a , Marc D. Green a , and Susan L. Forsburg a a Program in Molecular and Computational Biology, University of Southern California, Los Angeles, CA 90089; b Department of Chemistry and Biology, Ryerson University, Toronto, ON M5B 2K3, Canada http://www.molbiolcell.org/content/suppl/2015/08/03/mbc.E15-05-0318v1.DC1.html Supplemental Material can be found at: 3440 | S. A. Sabatinos et al. Molecular Biology of the Cell tants in other genes that bypass replication initiation do not repli- cate DNA but enter mitosis. This causes a cell untimely torn (cut) phenotype in which the unreplicated DNA is cleaved by the septum (e.g., cdc18∆, rad4ts, or orp1-ts; Kelly et al., 1993b; Saka and Yanagida, 1993; Grallert and Nurse, 1996). Presumably, initiation mutants never begin DNA replication and do not generate signals to trigger the checkpoint (Kelly et al., 1993a). We observe a novel phenotype in the temperature-sensitive mcm4-degron allele. This mutant has a degron cassette fused to the mcm4 temperature-sensitive (ts) allele to enhance protein turn- over (Lindner et al., 2002). Unlike the well-characterized mcm4-ts allele (cdc21-M68), Mcm4 degron protein is <10% of the original level during incubation at 36°C (Supplemental Figure S1A). We monitored DNA synthesis by nucleoside analogue incorporation (5-ethynyl-2′-deoxyuridine [EdU]) and detected very little accumu- lation in mcm4-degron at the restrictive temperature (Figure 1A and Supplemental Figure S1B). In contrast, mcm4-ts at 36°C incor- porates nearly wild type EdU amounts. Only early replication ori- gins fire in mcm4-degron, whereas mcm4-ts cells activate a combi- nation of early and late origins more efficiently (Supplemental Figure S1C). Surprisingly, despite these global replication defects, mcm4-degron cells divide multiple times at 36°C (Figure 1, B and C, and Supplemental Video S1), causing uneven DNA segregation in daughters and granddaughters. fork collapse generating DNA double-strand breaks. Cells with the mcm4-degron allele show DNA underreplication with accumulation of DNA damage markers at the restrictive temperature but are unable to maintain a checkpoint arrest. After release from replica- tion stress, these damaged cells continue to divide. The divisions are abnormal, producing ultrafine DNA bridges, multipolar spin - dles, and uneven chromosome segregation accompanied by the formation of small, satellite nuclei. These apparent micronuclei re- tain DNA damage markers and frequently rejoin with the parent nucleus. Significantly, the cells that survive this stress show substan - tially increased rates of mutation and chromosome rearrangement. This phenotype is distinct from mutants that block replication initia- tion, which fail to undergo chromosome segregation. Our data sug- gest that underreplication is a critical factor associated with genomic instability and establish a genetic model to investigate the links be- tween replication stress, disruptions in chromosome segregation, and genome rearrangements. RESULTS Most temperature-sensitive MCM helicase mutants duplicate the majority of their genome at 36°C (Figure 1A). However, these mcm- ts mutants accumulate DNA damage and trigger cell division cycle arrest (e.g., Nasmyth and Nurse, 1981; Coxon et al., 1992; Liang and Forsburg, 2001). On the other hand, temperature-sensitive mu- FIGURE 1: Underreplicated mcm4-degron mutants divide during and after replication stress. (A) EdU incorporation is lowest in mcm4-degron cells during incubation at 36°C. Asynchronous cultures were shifted to 36°C during EdU exposure, and synthesis was measured by EdU-FACS fluorescence in arbitrary units (A.U.). (B) Wild-type (wt) and mcm4-degron cells divide at least once at 36°C (6-h total videomicroscopy of asynchronous cultures at 36°C). Significantly more mcm4-degron cells undergo reductional anaphase (gray) in this first division at 36°C. Proportions in B, C, and E are shown with 95% CI, Z test of significance (*p < 0.01, **p << 0.001). (C) mcm4-degron daughter cells divide their DNA unevenly at 36°C, whereas wild-type daughters delay division at 36°C. Samples as in B, daughter cell divisions only. (D) Mutants incorporate less EdU after release at 25°C, as measured by EdU FACS. The initial asynchronous population was treated for 4 h at 36°C before release at 25°C in the presence of EdU. (E) Proportion of abnormal nuclear divisions in single-time-point images acquired before heat (asynchronous [A]), at 36°C (2, 4 h), and after 2 h of recovery at 25°C (R). (F) mcm4-degron cells during 25°C release with RPA-CFP (blue), Rad52-YFP (green), and histone-RFP (red). Unequal histone division in one cell (*) and a bridge (>) with a lagging chromosome and repair focus are common. Scale bar, 10 μm. Volume 26 October 1, 2015 Apparent yeast micronuclei in MCM mutant | 3441 Figure S1C) as Cnp1 foci scatter, indicating that centromeres repli- cate, separate, and possibly fragment. These multiple mitotic abnor- malities promote DNA missegregation after replication stress in mcm4-degron. We examined evidence for chromosome rearrangement using a lacO array near centromere I (Figure 2F). Many mcm4-degron cells failed to separate lacO Cen1 foci to both daughters, causing >2 green fluorescent protein (GFP) foci/nucleus or none at all. Because lacO arrays are potential fragile sites in Schizosaccharomyces pombe (Sofueva et al., 2011), a lacO Cen1 rearrangement or duplication may occur after mcm4-degron replication stress, causing cells with greater than two separating foci. This is also consistent with evi- dence for centromere fragmentation and rearrangement. Increased mutations and rearrangements in surviving mcm4-degron cells We next asked whether the 10% of surviving mcm4-degron cells show lasting signs of genome instability after transient replication stress (Figure 3A). We tested surviving cells for forward mutations that cause canavanine resistance (can1 +(S) to can1 −(R) ; Figure 3B). The baseline mutation frequency in mcm4-degron cells is higher than for wild type and mcm4-ts and significantly increases after in - cubation at 36°C. We also saw high rates of marker loss at other loci, including loss of an integrated marker at his7 (Supplemental Figure S3). To assess potentially catastrophic chromosome rearrangements, we introduced a nonessential minichromosome into the mcm4-ts and mcm4-degron strains. This minichromosome carries multiple genetic markers to maintain its overall stability or monitor its struc- tural integrity (Figure 3C). A low level of chromosome rearrange- ment is observed in wild-type cells, including break-induced replica- tion and isochromosome formation (Figure 3, D and E; Nakamura et al., 2008). Increased rearrangements are also observed in replica- tion fork protection complex mutants (Li et al., 2013). Consistent with the minichromosome maintenance (mcm) phe- notype, we observed an increased rate of minichromosome loss in both mcm4ts and mcm4-degron relative to wild type at 25°C (Figure 3E). Chromosome loss is modestly increased after incuba- tion at 36°C in the mcm4-degron strain but shows no significant change in the mcm4-ts background. Both mcm4-degron and mcm4-ts strains show an increased rate of rearrangement relative to wild type at 25°C (Figure 3D). However, after a 4-h pulse at 36°C, the mcm4-degron mutant shows a dra- matic increase in chromosome rearrangements that is not observed in mcm4-ts. Thus the division abnormalities observed in mcm4-de- gron are accompanied by increased mutations, chromosome rear- rangements, and chromosome loss. Damage persists in mcm4-degron mutants The ability of underreplicated mcm4-degron cells to divide repeat- edly suggests either that there is little DNA damage or that the damage checkpoint is not activated. To address the first point, we examined DNA repair proteins during arrest and release by visual- izing fluorescently tagged versions of the ssDNA-binding protein RPA, an early damage marker, and the Rad52 recombination pro- tein. The mcm4-ts mutant forms many discrete RPA and Rad52 foci during arrest at 36°C, which coalesce into a bright, pannuclear sig- nal upon release (Figure 4A and Supplemental Figure S4, A–C). This is consistent with earlier observations (Bailis et al., 2008) suggesting widespread late replication fork collapse at multiple sites, similar to the checkpoint mutant cds1∆ in HU (Sabatinos et al., 2012). In con- trast, mcm4-degron mutants form one or two large, distinctive RPA When cells are returned to 25°C, wild-type cells robustly con- tinue DNA synthesis and proliferation (Figure 1D and Supplemental Figure S1D). In contrast, neither mcm4-ts nor mcm4-degron cells incorporate much EdU after release to 25°C, a period that we call “recovery.” This low incorporation suggests that either there is lim- ited, residual synthesis across the genome or just a few cells re- turned to the cell cycle. The mcm4-ts cells remain cell cycle arrested after release, consistent with persistent DNA damage (Bailis et al., 2008). Surprisingly, mcm4-degron cells continue to divide (Supple- mental Figure S1E and Supplemental Video S2). Spindle pole body (SPB) duplication and separation occurs with timing similar to that for wild type (Supplemental Figure S1F). However, the segregation of the DNA in mcm4-degron is highly abnormal (Figure 1E), form- ing lagging chromosomes, replication protein A (RPA)–labeled ul- trafine bridges, and unequal DNA segregation into aneuploid and anucleate cells (Figure 1F and Supplemental Figure S1G). Apparent micronuclei form in underreplicated mcm4-degron cells We examined abnormal segregations in mcm4-degron more closely using live-cell video microscopy. The nuclear histone signal shows uneven segregation and fragmentation in >50% of mcm4-degron cells (Figure 2, A and B). These nuclear fragments form during mito- sis and are enclosed in separate nuclear membranes, which is the definition of a micronucleus (Hatch et al., 2013; Figure 2, A–C, Sup- plemental Figure S2, A and B, and Supplemental Video S3). The proportion of fragmented histone masses was similar to the number of membrane-bound micronuclei, indicating that a membrane ini- tially surrounds most wandering DNA fragments. There are no obvious connections between the micronucleus and the parent nucleus. Some membrane stalks remain indepen- dently attached to the septum (Supplemental Figure S2B). When these membrane-enclosed fragments remain in the same cell, they frequently rejoin the mother nucleus (∼60% of the time). Others seg- regate into a daughter cell during division, forming aneuploid cells. Subsequent divisions often show repeated segregation/fusion cycles (Supplemental Videos S4 and S5). Supplemental Video S4 shows delayed and failed mitosis followed by a later division, sug- gesting a dual spindle (e.g., Figure 2E and Supplemental Video S4). Thus these missegregations may also be linked to mitotic defects such as multipolar spindle formations. Another mitotic abnormality observed in mammalian cells after replication stress is ultrafine DNA bridges (UFBs) between fragile DNA regions (reviewed in Chan and Hickson, 2009). UFBs are not detected using DNA stains (e.g., 4′,6-diamidino-2-phenylindole [DAPI], histone; Chan et al., 2009) but can be visualized with RPA (Chan and Hickson, 2009). We observed twisting threads of RPA spanning unequal DNA masses in 20% of mcm4-degron divisions (Figure 2D, Supplemental Figure S2, C and D, and Supplemental Video S6). The RPA signal was often separate from the histone sig- nal, suggesting that single-strand DNA (ssDNA) has pulled apart from the bulk chromatin. These division anomalies resemble mitosis with unreplicated ge- nomes (MUGs), which happens in replication-arrested human cells that bypass the G2 damage checkpoint (Wise and Brinkley, 1997). One MUG characteristic is centromere fragmentation (Beeharry et al., 2013), which we detected in strains expressing a tagged cen- tromere-associated histone Cnp1–red fluorescent protein (RFP; CENPA homologue; Supplemental Figure S2E). Fission yeast centro- meres replicate early (Zhu et al., 1992) and then cluster with the SPB, except briefly during metaphase-to-anaphase transition. We ob - served early centromere replication in mcm4-degron (Supplemental 3442 | S. A. Sabatinos et al. Molecular Biology of the Cell filaments that extend from the center forming cups and voids that contain Rad52 (Figure 4B). At this higher resolution, the RPA/Rad52 foci are not simple dots but instead highly structured patterns within a 0.2-μm diameter. The 3D-SIM images show that Rad52 and RPA fit together end to end and that RPA tendrils loop out into surrounding histone regions (Supplemental Figure S4E). Further, we observe that the megafocus occurs in histone-deficient nuclear regions (Supple - mental Video S7). “megafoci.” RPA and Rad52 colocalization in both mcm4 mutants (Supplemental Figure S4D), coupled with their low viabilities, suggests that these are dominated by stalled or damaged replica- tion forks (e.g., Lambert et al., 2010) and not stably stalled replica- tion forks (Irmisch et al., 2009). We used superresolution microscopy to examine the megafocus substructure. Three-dimensional (3D) structured illumination micros- copy (SIM) images show that the megafocus is an RPA complex, with FIGURE 2: Underreplication followed by division promotes micronuclei and genomic rearrangement in fission yeast. (A) mcm4-degron cells released to 25°C (after 4 h, 36°C) form anucleate cells (*) and apparent micronuclei (>). Histone- RFP and membrane (ccr1N-GFP) are shown; scale, 10 μm. Micronuclei are often resorbed back into the main nucleus (time 1:15–1:45 h:min) after release to 25°C. (B) Chromatin (histone-RFP) frequently separates into discrete, condensed fragments that are separate from the main nuclear mass in mcm4-degron cells. More than 50% of these fragments rejoin the parent nucleus (PN). Asynchronous cultures were treated (4 h, 36°C) and then shifted to 25°C for microscopy during recovery (12-h cumulative data, two or three biological replicates per strain). The proportion of cells that form separate histone bodies is shown relative to total number of cells monitored. (C) Membrane-enclosed chromatin masses frequently separate from the main nucleus in mcm4-degron cells but are rarely detected in wild type (wt). More than 60% of these micronuclei fuse and rejoin the parent nucleus (PN) during recovery at 25°C (12-h cumulative data, two to four biological replicates per strain). (D) mcm4-degron cells develop dynamic ssDNA (RPA-CFP , blue) bridges dotted with Rad52-YFP (yellow) during release at 25°C (time in hours:minutes). (E) Evidence for two spindles resulting in apparent micronuclei in some mcm4-degron cells during recovery (after 4 h, 36°C). Conditions as in A; scale, 10 μm. (F) A LacO array near the centromere 1 (lys1+-lacO Cen1 ) unevenly separates in mcm4-degron divisions after 4 h at 36°C. More than two dots are frequently observed in mcm4-degron, which suggests that the array is rearranged or fragmented. LacI-GFP (green) bound to lacO Cen1 is shown below relative to DNA signal (histone-RFP). Stacked histogram for pooled data from three biological replicates, with chi-squared test of significance for proportion of single dots (gray) or more than three dots (black) segregating (**p << 0.001). Volume 26 October 1, 2015 Apparent yeast micronuclei in MCM mutant | 3443 marks replication origins (orp1-4; Grallert and Nurse, 1996), and Rad4 TopBP1 is essential for replication initiation and also activation of the DNA damage checkpoint (rad4-116; Saka and Yanagida, 1993). These mutants enter a lethal mitosis with unreplicated DNA that is cleaved by the cell septum (cut). Both orp1ts and rad4ts formed some RPA and Rad52 foci, but the quantity and patterns were differ- ent from those for mcm4-degron (Supplemental Figure S5, A–C). The rad4ts mutants are much shorter at division, typical of cut mu- tants, with a sub-1C DNA content and increased cell death (Supple- mental Figure S5, D and E). Fewer chromosome missegregations occur in either orp1ts or rad4ts than in mcm4-degron, particularly during recovery at 25°C (Supplemental Figure S5F). Thus mcm4- degron defines a new class of early replication mutant. mcm4-degron transiently activates the damage checkpoint and then escapes RPA contributes directly to fork stability and damage checkpoint activation (Zou et al., 2003; Toledo et al., 2013). The checkpoint kinase, Chk1, is phosphorylated in asynchronous mcm4-degron cells. Chk1 activation, detected by a band shift Western blot The presence of Rad52 repair foci in ∼15% of untreated mcm4- degron cells suggests that the cells suffer damage even under permissive conditions. Pulsed-field gels show that untreated mcm4- degron chromosomes migrate poorly and generate a low–molecular weight smear indicating DNA breaks (Supplemental Figure S4F). This genome instability may be due to Mcm4 degron protein instability compared with wild-type Mcm4 before temperature shift (Supple- mental Figure S1A). These observations are consistent with our pre- vious observation that reduced MCM levels cause genome instabil- ity before replication is noticeably affected (Liang et al., 1999). Surprisingly, RPA and Rad52 foci persist in dividing mcm4-de- gron cells (Figure 4C and Supplemental Videos S2 and S8). We also see RPA and Rad52 foci in the apparent micronuclei (Figure 4D); these may be markers of ongoing DNA synthesis, stalled forks, or DNA damage. Consistently, we find that these signals appear later in the putative micronuclei than in the primary nucleus (Figure 4D, arrowhead vs. asterisk in the primary nucleus) and can be reincorpo- rated into the parent nucleus (Figure 4E). The phenotypes we observe with mcm4-degron are different from those seen in other replication initiation mutants. Orp1 ORC1 FIGURE 3: Transient replication instability causes mutation in surviving mcm4-degron cells. (A) Relative viability of cultures at 36°C (strains FY4743, FY4857, FY5279). Cells were shifted to 36°C and plated at time points to determine viability relative to the starting culture. (B) Mutation rate (can1 + ) increases in mcm4-degron after 4 h at 36°C (n = 7). *p < 0.001 comparing wt or mcm4-ts with mcm4-degron; °p << 0.001 change from 25 to 36°C in mcm4-degron. Plots in B, D, and E show a center median line bounded by 25th and 75th percentiles. (C) Schematic for the minichromosome (ChL) assay, followed by markers (also see Nakamura et al., 2008; Li et al., 2013). Cells may lose ChL or undergo gross chromosomal rearrangements (GCRs). An isochromosome (ChL-iso) is formed by duplication of the left arm with the LEU2+ marker producing a smaller chromosome. Break-induced replication (BIR) products may occur between ChL and chromosome III, producing a longer product that is frequently hygromycin resistant. (D) GCR events are highest in mcm4-degron after 4 h at 36°C. Significant median differences from wild type at 25 or 36°C are reported as p < 0.001 (*) with outliers (o). (E) ChL loss is highest in mcm4-degron after 4 h at 36°C and even before replication stress at 25°C. Loss is also higher in mcm4-degron compared with mcm4-ts (p < 0.02. all conditions). Conditions and analysis as in D. 3444 | S. A. Sabatinos et al. Molecular Biology of the Cell Consistent with these data, the inhibitory Cdc2 phosphorylation on threonine 15 that prevents mitosis (O’Connell et al., 1997) is reduced in mcm4-degron (Figure 5C). This checkpoint activation explains the robust cell cycle arrest of the mcm4-ts compared with mcm4-degron. Moreover, under these conditions, we Crb2 53BP1 levels drop sharply in mcm4-degron at 36°C, suggest- ing that the checkpoint signal is interrupted upstream of Chk1 (Figure 5B). (Figure 5B), is required for both checkpoint initiation and mainte- nance (Latif et al., 2004). In mcm4-degron, activated Chk1 is present in asynchronous cells but decreases at 36°C during rep- lication stress (Figure 5B) even as RPA and Rad52 foci form (Figure 4C). After release to 25°C, Chk1 is moderately phosphor- ylated in mcm4-degron, and cells continue to divide. In contrast, Chk1 is inactive in asynchronous mcm4-ts cells but becomes highly phosphorylated at 36°C and during release at 25°C. FIGURE 4: Divisions occur in the presence of DNA damage and repair signals. (A) RPA focus patterns during replication collapse are different in each mutant but rarely develop in wild type at 36°C (i). Multiple (more than three) punctate RPA foci form in mcm-ts nuclei after 4h 36°C (ii) and later become a pannuclear RPA signal like that observed in cds1∆+HU (iv). A unique “megafocus” of bright, compact RPA forms in mcm4-degron (iii). Heat map scale (top) and 2- μm scale. (B) 3D-SIM images of mcm4-degron nucleus after 4 h 36°C (top left) in one midfocal z-section (xy); scale bar, 2 μm. (i, ii) Enlarged yz-perspectives of surface-rendered megafocus. Also see Supplemental Video S5. (C) RPA and Rad52 foci are present at division in mcm4-degron (also see Supplemental Videos S2 and S6). (D) DNA damage (RPA-CFP , top magenta) and DNA repair foci (Rad52-YFP , bottom magenta) develop in newly formed micronuclei (MN; assessed with membrane marker ccr1N-GFP; green). Cells were incubated at 36°C, 4 h before videomicroscopy during release at 25°C for 6 h. A time scale is indicated on the bottom right corner of each panel (hours:minutes). The MN form damage and repair signals after they are first detected in the parent nucleus (*, parent; >, MN), before rejoining. Scale bar, 5 μm. (E) A proposed model for transient replication-stress inducing mutations in surviving cells after mcm4-degron inactivation, shown at the level of the nucleus (nuclear membrane in blue). Cells treated 4 h at 36°C are underreplicated (step 1) but divide, causing UFBs and fragmented DNA during mitosis (step 2). Fragments are membrane-bound MN that develop DNA damage (step 3). Resorption of MN back into the parent nucleus promotes further genome instability during 25°C recovery, leading to the development of a mutated surviving population (step 4). Volume 26 October 1, 2015 Apparent yeast micronuclei in MCM mutant | 3445 DISCUSSION Mcm4 is an essential subunit of the MCM helicase, the primary rep- licative helicase of eukaryotic cells (e.g., Maiorano et al., 2000; Labib et al., 2001; Ishimi et al., 2003a; Yabuta et al., 2003). Disrupting Mcm4 function drives genome instability in many models. Mouse mcm4 mutations are associated with chromosome breaks, genome rearrangements, micronucleus formation, and breast or blood can- cers (Shima et al., 2007a,b; Bagley et al., 2012). It has been pro- posed that this reflects a failure to license additional replication ori - gins that allow rescue of a failed replication fork (Kawabata et al., 2011; McIntosh and Blow, 2012). In humans, MCM4 truncation mu- tations are associated with chromosome instability and DNA repair defects (Casey et al., 2012; Gineau et al., 2012; Hughes et al., 2012). MCM overexpression is correlated with hyperproliferation and carci- nogenesis (e.g., Ishimi et al., 2003b; Guida et al., 2005). Therefore Mcm4 plays a fundamental role in maintaining genome stability. We characterized a novel temperature-sensitive allele of mcm4 in fission yeast (mcm4-degron) that generates a distinct form of early replication stress in which early replication forks fire but un - dergo little DNA synthesis. This is accompanied by transient DNA damage checkpoint activation and then escape, suggesting that cells are unable to initiate or maintain a checkpoint response (Latif et al., 2004). Because there are low levels of checkpoint mediator protein Crb2 at 36°C, the checkpoint activation step in mcm4- degron may not be amplified (Lin et al., 2012); this agrees with re- cent work proposing that Chk1 activation is linked to the MCM com- plex (Han et al., 2014). Alternatively, mcm4-degron checkpoint maintenance might fail, allowing escape, as is the case at telomeres where Crb2 is absent (Carneiro et al., 2010). This contrasts with mcm4-ts mutants, which synthesize almost a wild-type amount of DNA before undergoing robust checkpoint- dependent arrest. We observe that Mus81 endonuclease is required Without Chk1, the mcm-ts chk1∆ double mutants enter prema - ture mitosis and cut at 36°C (Supplemental Figure S6, A and B; Liang et al., 1999). In mcm4-degron chk1∆ double mutants, the fraction of cut cells is only slightly higher than in mcm4-degron alone. This suggests that the Chk1 checkpoint transiently restrains division in mcm4-degron at 36°C. Once returned to 25°C, there is no difference between division numbers and morphology in mcm4- degron and mcm4-degron chk1∆ double mutants. Mus81 promotes checkpoint arrest during late-replication failure What is different about the late replication fork failure in mcm4-ts and the early collapse in mcm4-degron? Whereas mcm4-ts accumu- lates DNA breaks and robustly activates the damage checkpoint, mcm4-degron does not. The contrast in their RPA patterns and tim- ing suggests that the two mutants generate different replication ar- rest structures. Because Mus81 endonuclease reportedly cleaves stalled replication forks in late S phase to promote fork restart (Froget et al., 2008; Saugar et al., 2013), we reasoned that Mus81 might cleave mcm4-ts arrested forks to form DNA breaks and gen- erate a robust damage signal Consistent with this model, we found that a mcm4-ts mus81∆ double mutant showed a dramatic increase in dividing cells at 36°C compared with mcm4-ts alone (Figure 5D) and thus resem- bles the mcm4-degron. In contrast, mus81∆ did not change the proportion of mcm4-degron cells forming micronuclei or under- going asymmetric divisions during release (Supplemental Figure S6, C–E). We infer that Mus81-dependent damage formed in mcm4-ts generates a signal for robust G2 checkpoint activation and cell cycle arrest. In contrast, the early-failing replication forks in mcm4-degron fail to activate fully or maintain the G2 checkpoint. FIGURE 5: Underreplication promotes micronuclei, DNA damage, and aneuploidy in fission yeast. (A) Experimental scheme. Asynchronous cells were shifted to 36°C for 4 h total and then released to 25°C for 2 h. (B) The DNA damage checkpoint becomes activated by Chk1-HA phosphorylation (*) in methyl methanesulfonate (MMS)-treated wild-type cells (+M) and in mcm4-ts. Chk1-HA is moderately phosphorylated in asynchronous mcm4-degron and never attains activated levels of mcm4-ts, as assessed by the ratio of modified (top) to unmodified (bottom) Chk1. The 53BP1 homologue Crb2 is phosphorylated in response to MMS treatment and stable in wild type but is rapidly lost in mcm4-degron at 36°C. Arrowheads (<) indicate modified forms of proteins, and the bar (–) indicates a non–HA-tagged control lysate. (C) Cdc2 is not phosphorylated in mcm4-degron at 36°C and only minimally during recovery (25°C). In contrast, high-level, sustained Cdc2 phosphorylation occurs in mcm4-ts. Cdc2 modified and unmodified protein levels were detected on Western blots and quantified to plot the ratio at each time point. (D) Loss of Mus81 endonuclease (mus81∆ ) increases divisions in mcm4-ts mus81∆ , forming aneuploid and cut cells. 3446 | S. A. Sabatinos et al. Molecular Biology of the Cell evidence that chromosomes within micronuclei are severely dam- aged or pulverized (Kato and Sandberg, 1968; Crasta et al., 2012; Zhang et al., 2015). The resulting chromosome rearrangements may be incorporated into the genome if the micronuclear DNA merges with the parent nucleus during mitosis (reviewed in Forment et al., 2012; Holland and Cleveland, 2012; Zhang et al., 2013). These ob- servations have led to the suggestion that aberrant micronucleus segregations are associated with the catastrophic chromosome re- arrangements termed chromothripsis (Crasta et al., 2012; Holland and Cleveland, 2012; Zhang et al., 2015). The events that generate micronuclei are likely linked to other cytogenetic abnormalities, including chromosome bridging, break- age-fusion-bridge cycles, and centromere fission (e.g., Fenech et al., 2011; Martinez and van Wely, 2011; Sorzano et al., 2013). In mammalian cells, caffeine-induced checkpoint bypass produces evi- dence of centromere fragmentation in underreplicated cells (Burrell et al., 2013). Centromere breaks and fission have been associated with micronucleus formation and chromosome rearrangements (Guerrero et al., 2010; Martinez and van Wely, 2011). Consistent with this, we previously described the fission yeast pericentromere repeats as vulnerable to rearrangement during replication stress (Li et al., 2013). The unusual mcm4-degron mutant phenotype estab- lishes a yeast model to examine missegregation events in which we observe evidence for centromere fission, UFBs, and abnormal/ aneuploid segregation. We predicted that these abnormal segregations and apparent micronuclei should be associated with increased evidence of ge- nome instability, and genetic studies showed this to be the case. The mcm4-degron strain is a mutator, with increased accumula- tion of forward mutations after incubation at 36°C. Using a nones- sential minichromosome (e.g., Nakamura et al., 2008; Li et al., 2013), we observed a striking increase in chromosome rearrange- ments in the mcm4-degron cells that survive replication stress compared with wild-type or mcm4-ts cells, which maintain check- point arrest. We infer that the abnormal divisions of mcm4-degron establish a source of continuing genome instability (model in Figure 4D). A frac- tion of the underreplicated genome is separated during mitosis and shows accumulation of RPA and Rad52 foci later than in the parent nucleus. This could reflect DNA damage or asynchronous DNA rep- lication. Apparent nuclear fusion or rejoining between the sepa- rated body and the parent nucleus reincorporates the damaged DNA into the parent nucleus after mitosis. We hypothesize that this is one cause of enhanced mutation rate after transient mcm4- degron inactivation. Intriguingly, data in mammalian systems sug- gest that DNA damage that occurs during mitosis can be masked until the next cell cycle (e.g., Lukas et al., 2011). Of importance, we show that transient replication instability has long-reaching effects and that genome instability (persistent RPA/Rad52 foci, bridges, and apparent yeast micronuclei) is established and transmitted over multiple divisions during growth reestablishment (Supplemental Videos S4 and S5). Of course, nuclear membrane dynamics differs in yeast and mammals. We observe that ∼70% of fission yeast micronuclei fuse with the parent nucleus. This is similar to the frequency observed for micronuclear DNA rejoining the parent DNA during mitosis in mi- crotubule-destabilized mammalian cells (Hatch et al., 2013). How- ever, micronuclear membrane fusion is not reported in mammalian cells (Crasta et al., 2012). The open mammalian mitosis, with nuclear envelope breakdown, allows micronuclear DNA to rejoin the parent nucleus when the nuclear membrane is degraded during mitosis. In contrast, the fission yeast mitosis is closed, and the nuclear envelope to maintain activation of Chk1 in mcm4-ts cells. This suggests that Mus81 recognizes and acts upon a specific structure formed during late fork collapse in mcm4-ts, and this generates the robust check- point signal that maintains cell cycle arrest. This may be due to inac- tivation of Mus81 during early S phase, as is observed in Saccharo- myces cerevisiae (Saugar et al., 2013). Alternatively, there may be no Mus81-susceptible substrates formed in mcm4-degron early repli- cation arrest, preventing a strong G2 checkpoint activation. The mcm4-degron cells show replication stress even without a temperature shift, as indicated by their constitutive DNA repair foci (Rad52), smeared chromosomes by pulsed-field gel electrophoresis (PFGE) analysis, higher mutation rates, and constitutively activated Chk1. This is consistent with other work showing that reduced MCM protein levels contribute to genome instability (Liang et al., 1999; Gineau et al., 2012). The mcm4-degron cells also acquire a novel RPA/Rad52 structure that is not seen in other replication-initiation mutants. We propose that this “megafocus” represents early-firing replication origins that are clustered during initiation (Knott et al., 2012) and then collapse as Mcm4 degron protein is lost. Our super- resolution analysis of the mcm4-degron megafocus shows that the structures of ssDNA and Rad52-bound DNA are intertwined. These megafoci of colocalized RPA and Rad52 do not stably activate the DNA damage checkpoint, similar to replication stress–induced foci of brc1∆ mutants (Bass et al., 2012). In our model, Mcm4 degron preas- sembled at early origins is protected from immediate inactivation at 36°C, and the protein becomes vulnerable during the transition to replication elongation, causing replication failure and ssDNA accu- mulation at an early stage. In contrast, the mcm4-ts mutants arrest with numerous dispersed RPA foci, consistent with late fork collapse detected by phosphorylated H2A(x) (Bailis et al., 2008). This may occur stochastically or at specific fragile sites. Unexpectedly, we observe that despite underreplication, most mcm4-degron cells divide during both replication stress at 36°C and again after release (Figures 2 and 4). These abnormal mitoses pro- duce UFBs marked with RPA and apparent centromere fragmenta- tion. Cells undergo continued, abnormal divisions that generate small, membrane-bound bodies that contain a subset of the ge- nome. These may segregate into separate daughter cells, generat- ing aneuploidy, or remain in the mother cell, where they may rejoin the parent nucleus. These structures are intriguingly suggestive of micronuclei. In mammalian cells, micronuclei form when a subset of the ge- nome is separated into distinct membrane-bound bodies. These may form after irradiation (e.g., Kato and Sandberg, 1968) or when cells with replication defects enter mitosis (Kato and Sandberg, 1968; Shima et al., 2007a; Chan et al., 2009; Utani et al., 2010; Bagley et al., 2012). Micronuclei may contain acentric genome frag- ments or whole chromosomes and may be associated with dicen- trics and chromosome bridges. These data indicate that they may result from different forms of genetic stress or mitotic failure (e.g., Fenech et al., 2011). Although micronuclei are common markers in cancer cells (e.g., Crasta et al., 2012; Hatch et al., 2013), the relationship between their formation, stability, and overall genome instability is not under- stood. For example, micronuclei clearly form in response to whole- genome damage and replication stress, as seen in mouse Mcm4 mutants (Shima et al., 2007a; Gineau et al., 2012), and yet spindle poisons that perturb mitosis also cause whole-chromosome misseg- regation and micronuclei (Crasta et al., 2012; Hatch et al., 2013; Zhang et al., 2015). In the latter studies, DNA replication is delayed in micronuclei compared with the parent nucleus (Crasta et al., 2012), leading to DNA damage. Indeed, there is long-standing Volume 26 October 1, 2015 Apparent yeast micronuclei in MCM mutant | 3447 were used, and numbers were pooled. The combined proportions with 95% CI are presented. Deconvolved and projected images from a time course are shown in Figure 2A. A projected image of a single cell is shown in Supplemental Figure S2 and Supplemental Video S3. Protein methods Protein extracts were prepared from equal numbers of cells treated with 0.3 M sodium hydroxide. Cells were lysed by boiling for 5 min in acidic SDS–PAGE buffer (4% SDS, 60 mM Tris-HCl, pH 6.8, 5% glycerol, 4% 2-mercaptoethanol, 0.01% bromophenol blue, 0.1 M dithiothreitol). Samples were run on Tris-glycine gels and transferred to polyvinylidene fluoride membrane. Primary an- tibodies for Chk1HA (16B12 anti-hemagglutinin [HA]; Covance), phospho–Cdc2-Y15 (Cell Signaling Technology, Danvers, MA), and S. pombe Cdc2, Mcm4, and Crb2 (polyclonal antibodies) were incubated overnight. Blots were washed in phosphate-buff- ered saline (PBS)–Tween buffer, exposed to horseradish peroxi- dase–conjugated secondary antibody for 1 h, and then washed and exposed using enhanced chemiluminescence (Pierce). Quan- titation of Mcm4 and Chk1-HA (phosphorylated and unmodified forms) was performed using QuantityOne software (Bio-Rad) as in Furuya et al. (2010). DNA synthesis detection To monitor DNA synthesis by nucleoside analogue incorporation, cultures were treated with either 10 μM EdU or 10 μg/ml bromode- oxyuridine (BrdU) for appropriate times before harvest. EdU-treated cells were fixed with 70% ethanol and processed using the Click-iT EdU Alexa Fluor 488 Imaging Kit according to directions (C10337; Life Technologies, ThermoFisher Scientific). BrdU chromatin immu- noprecipitation (IP) was performed as described in Knott et al. (2012) with the following modifications. Cells were pelleted, snap- frozen, and then stored at −80°C. After lysis in TES (100 mM Tris, pH 8.0, 50 mM EDTA, 1% SDS) with glass beads, chromatin was sheared by sonication, resulting in ∼500–base pair fragments. DNA was phenol-chloroform extracted, isopropanol precipitated, and then resuspended in TE. Samples were diluted with IP buffer (1× PBS, 0.05% Triton X-100) before overnight incubation with anti-BrdU (RPN202; GE Healthcare, Sigma-Aldrich). Antibody-BrdU- DNA complexes were precipitated on magnetic protein A–Sepha- rose (Dynabeads, 10002D; Invitrogen, ThermoFisher Scientific), washed three times in IP buffer and once in TE, and then incubated in TES at 65°C (15 min). DNA was then purified using a Qiagen PCR purification kit and quantitatively amplified on a PerkinElmer HT9700 using origin-specific primers (Supplemental Table S2) and iTaq Universal SYBR Green Supermix (Bio-Rad, Hercules, CA). The Pfaffl method was used to determine percentage IP for each region relative to input DNA. Flow cytometry and microscopy Cells were fixed in cold 70% ethanol for cell cycle analysis or micros - copy. For DAPI/septa staining, cells were rehydrated in water and incubated for 10 min in 1 mg/ml aniline blue (M6900; Sigma- Aldrich). Cells in mount (50% glycerol, 1 μg/ml DAPI, and 1 μg/ml p-phenylenediamine) were photographed on a Leica DMR wide- field epifluorescence microscope using a 63 × objective lens (numer- ical aperture [NA] 1.62 Plan Apo), 100-W Hg arc lamp for excitation, and a 12-bit Hamamatsu ORCA-100 charge-coupled device (CCD) camera. OpenLab version 3.1.7 (ImproVision, Lexington, MA) soft- ware was used at acquisition and ImageJ (National Institutes of Health, Bethesda, MD) for analysis. does not degrade. Therefore the mechanism of yeast micronuclear DNA rejoining may be different and appears to be postmitotic. Of importance, the abnormal segregation we observe is clearly mitotic in origin. It is led by centromeres and spindle pole bodies (Figure 2E and Supplemental Figure S2E) and is distinct from abnormal nuclear budding, such as that observed in fission yeast mutants with dis - rupted nuclear membrane dynamics (Sazer et al., 2014). This is the first report of micronucleus-like divisions in fission yeast, and it is not observed in the other early replication mutants tested (orc1ts, rad4ts). Thus these divisions are a feature of a very specific early replication defect that allows some fraction of the ge - nome to undergo segregation, evading the checkpoint by circum- venting DNA breakage through Mus81. Micronuclei induced by a yeast mcm4 mutation are particularly intriguing, given the association of micronuclei, chromosome breaks, and cancers in mouse Mcm4 mutants (Shima et al., 2007a; Bagley et al., 2012). It is possible that disruptions in the MCM4 sub- unit are particularly linked to damage that evades the checkpoint and promotes abnormal mitosis. Significantly, yeast genetic tools now allow a detailed investigation of contributing factors and de- scription of outcomes. This provides a powerful genetic model to investigate the mechanisms of aberrant segregation and micronu- cleus formation caused by replication instability and the potential of large-scale genetic damage. MATERIALS AND METHODS Cell growth and physiology Fission yeast strains are described in Supplemental Table S1 and were grown as in Sabatinos et al. (2012). Physiology experiments for viability, DNA synthesis, Chk1 protein, PFGE, and flow cytometry were performed in supplemented Edinburgh minimal medium (EMM). Live-cell imaging cultures were grown in fully supplemented EMM with 5 μM thiamine and photographed in the same medium. Septation and nuclear counts were performed on fixed samples. Briefly, cells were fixed in 70% ethanol, rehydrated, and then stained in 1 mg/ml aniline blue (M6900; Sigma-Aldrich) for 15 min. Stained cells were mounted on glass slides with SlowFade Gold antifade mount with DAPI (S36938; Invitrogen, ThermoFisher Scientific) and photographed. More than 200 cells were counted from two biologi- cal replicates and pooled, and then proportions and 95% confi - dence intervals (CIs) were calculated. Differences in proportions were assessed with a two-tailed Z test. Micronucleus measurement An initial assessment of the micronucleus-forming potential in cul- tures was made by incubating cultures at 36°C for 4 h and then im- aging over 12 h at 25°C. Using a process similar to that of Hatch et al. (2013; Figure 1C), we monitored histone-RFP (hht1-RFP) in cells resolving division. The presence of smaller chromatin bodies away from the primary nucleus was scored as a “free chromatin body”; these were monitored to determine whether they rejoined the primary nucleus (resorbed). To determine whether free chromatin bodies were membrane- enclosed micronuclei, the membrane marker ccr1 N-terminal frag- ment (ccr1N-GFP) was monitored with histone (hht1-RFP) in live cells after 4 h at 36°C. Cells were scored as micronucleus forming if they met three criteria: 1) the micronuclear histone mass was sur- rounded by membrane and separated from the parent nucleus; 2) the micronucleus formed after nuclear division, excluding rare spontaneous micronuclei; and 3) if micronuclei were retained after septation, to exclude fragmented bodies that formed transiently during mitosis. Videos from more than two biological replicates 3448 | S. A. Sabatinos et al. Molecular Biology of the Cell REFERENCES Bagley BN, Keane TM, Maklakova VI, Marshall JG, Lester RA, Cancel MM, Paulsen AR, Bendzick LE, Been RA, Kogan SC, et al. (2012). A domi- nantly acting murine allele of mcm4 causes chromosomal abnormalities and promotes tumorigenesis. PLoS Genet 8, e1003034. 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Live-cell videomicroscopy experiments at 36°C were per- formed on 2% agarose pads sealed with VaLaP (1/1/1 [wt/wt/wt] Vaseline/lanolin/paraffin). Long-term videomicroscopy at 25°C was performed in CellAsics microfluidics plates (Y04C series; EMD Millipore), with constant temperature and medium flow. Fluores - cent-tag images of live cells were acquired using a DeltaVision mi- croscope (with softWoRx version 4.1; GE, Issaquah, WA) using a 60× (NA 1.4 PlanApo) lens, solid-state illuminator, and 12-bit CCD camera. Sections of static time points were eighteen 0.3-μm z-sec- tions. Long-term time-lapse videos used nine z-steps of 0.5 μm. Images were deconvolved and maximum intensity projected (soft- WoRx). Transmitted light images were added to projected fluores - cence images. Images were contrast adjusted using an equivalent histogram stretch on all samples. A threshold of signal 2× over the average nuclear background was used for RPA-CFP and Rad52- YFP focus discrimination. Foci are presented as the proportion of nuclei per category of focus with ±95% CI. Significance was as - sessed with chi-squared tests and differences between proportions with two-tailed Z tests. Mutation analysis The forward canavanine mutation rate at can1 + was determined as described (Sabatinos et al., 2013). Briefly, cultures were diluted in yeast extract with supplements (YES) medium and plated on 15-cm canavanine plates (70 μg/ml in pombe minimal medium with gluta - mate [PMG] plus supplements plus phloxine B). Plates were scored after 8 d at 25°C, for the number of can1 − colonies compared with total cells plated, calculated from titer plates. Grouped experiments were performed independently, and then the mutation rate was cal- culated using the MSS-MLE algorithm in FALCOR (www.keshavsingh .org/protocols/FALCOR.html). Results were plotted in Delta Graph and compared by two-tailed t test. The frequency of hsv-tk + loss and sectoring was scored in 500– 2000 cells plated on YES, grown at 25°C, and then replica plated onto fluorodeoxyuridine (FUdR) plates (20 μg/ml in EMM plus supplements plus phloxine B). The number of FUdR-resistant and sectored colonies was counted per total number of colonies and the proportions assessed with Z tests (vassarstats.net/propdiff_ ind.html). Grouped experiments were performed independently and pooled for analysis. A box plot of sectored data was made using BoxPlotR, showing median, 25th/75th percentile boundar- ies, and 1.5× interquartile whiskers (boxplot.tyerslab.com/). The ChL minichromosome strains were grown as in Nakamura et al. (2008) and Li et al. (2013). Cultures were plated for viability and on PMG-HULA plates with 5-fluoroacetate (5-FOA; Zymo Research, Irvine, CA). Cultures were then incubated at 36°C for 4 h and then plated as at the start. All plates were grown at 25°C, and the number of Ura– colonies counted and compared with the total number of surviving cells. Ura– colonies were replica plated onto PMG-HUA and PMG-HUL with 5-FOA to assess Ura– Leu– and Ura– Ade– colonies, respectively. Ura– Leu– colonies were patched or replica plated onto PMG-HUL and YES-hygromycin to assess hygromycin and Ade status as in Li et al. (2013). FALCOR ACKNOWLEDGMENTS We thank the University of Southern California Center for Electron Microscopy and Microanalysis for support with 3D-SIM microscopy and Stephen Kearsey, Jian Qu Wu, Xie Tang, and Zac Cande for strains. This work was supported by National Institutes of Health Grants R01 GM081418 and GM111040. was used to calculate recombination/mutation rates, and a Mann–Whitney two-tailed U test was used to assess significance between observed sets. Volume 26 October 1, 2015 Apparent yeast micronuclei in MCM mutant | 3449 Liang DT, Forsburg SL (2001). 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Forsburg 1 1 Program in Molecular and Computational Biology, University of Southern California, 1050 Childs Way, RRI 201B, Los Angeles, CA, 90089-2910 2 Department of Chemistry and Biology, Ryerson University, 350 Victoria Street Toronto, Ontario, CANADA M5B 2K3 Supplemental Files Include: Supplemental Figures S1 - S6 Supplemental Tables S1, S2 Supplemental Videos S1 - S8 Supplemental References (4) Figure S1, related to Figure 1. Characteristics of mcm4-degron and mcm4-ts mutants during temperature shift and release. A) Lysates from asynchronous cultures (AS) shifted to 36°C (2h, 4h), and then released to 25°C for 2h (R). Mcm4 antibody was used to detect protein levels relative to beta-tubulin, and normalize Mcm4 protein to total protein at each timepoint. Mcm4 protein level was calculated relative to starting amount in each sample during the experiment. Right, arrow indicates larger Mcm4 degron protein generated by insertion of degron cassette. B) FACS profiles of bulk DNA content (SytoxGreen) and EdU (bottom, log scale) during asynchronous shift to 36°C, related to Figure 1A. Plotted values of Fig 1A were taken from the median EdU peak values. C) Early origins are BrdU-enriched at 36°C (ori2004, CEN(dg)), but mcm4-degron late origins (ori3002, ori2080, ori727) fire less efficiently. BrdU incorporation is percent precipitated (%IP) relative to no IP control. In contrast, mcm4-ts activates early origins and several later origins more than detected for mcm4-degron (ori2080, ori727) D) As in S1B, FACS profiles of cells during release at 25°C (post-36°C, 4h), related to Figure 1D. E) Wild type and mcm4-degron cells, but not mcm4-ts, divide after release to 25°C (12h, cumulative numbers). mcm4-degron divisions are largely reductional anaphase. Proportion of dividing cells relative to total assessed (indicated below genotype) is shown with 95%CI, *p<<0.001. F) Spindle pole body (SPB) duplication, rotation and separation monitored in cultures that were treated for 4h 36°C and released at 25°C. As in Fig A1E, numbers are from 12h live-cell observation (cumulative) with analysis as in E. G) Separated damage and repair signals in the dividing cells image of Fig. 1f, forRad52-YFP (green), RPA-CFP (blue), and a merged image including histone (hht1-RFP, red). The transmitted light image of cell outlines is shown at far right. Scale 10 µm. Figure S2, Related to Figure 2. Chromosome mis-segregation produces DNA fragments, bridges and centromere separation. A) Chromatin (histone-RFP) frequently separates into discrete, condensed fragments that are separate from the main nuclear mass in mcm4-degron cells. Over 50% of these fragments rejoin the parent nucleus (PN). Asynchronous cultures were treated (4h, 36°C) and then shifted to 25°C for microscopy during recovery (12h cumulative data, 2-3 biological replicates per strain). The proportion of cells that form separate histone bodies is shown relative to total number of cells monitored. B) Related to Video 3, rotational projections of a mcm4-degron micronucleus and parent nucleus using ccr1N-GFP (green, membrane marker) and histone-RFP (magenta). Cells were treated at 36°C for 4h, then imaged at 25°C. The micronucleus is on a separate membrane projection and completely and separately encapsulated away from the primary nucleus. The micronucleus tether connects to the septum and not the primary nucleus. The membranes around the parent and micronucleus are intact, and evident dIsjunctions between the primary nucleus and the micronuclear membrane “stalk” are indicated by (>). C) Histone-RFP and RPA-CFP signals separate during division, and are transiently apart from each other. Data from cells after 4h 36°C, and released at 25°C for 12h recovery and constant live-cell imaging. D) Dynamic, RPA-coated ultra fine bridges (UFBs) are detected in approximately 20% of mcm4- degron divisions after 4h 36°C (release at 25°C, 12h total time). E) Centromeres (green, cnp1-GFP) frequently separate into more than 6 dots (>) during interphase in mcm4-degron (*). Compare to single dots in wild type interphase and separated (i). Cells at 25°C after 4h 36°C, shown with ssDNA signal (magenta, RPA-CFP). Figure S3, related to Figure 3. Evidence for mutation at other loci. A) Sectoring in mcm4-degron FUdR-sensitive colonies increases after 4h, 36°C, but not in wild type. Inset, examples of resistance (arrow), and sectored colonies in mcm4-degron after 4h 36°C. Sectoring indicates sporadic hsv-tk + loss and genome plasticity during colony growth. Figure S4, related to Figure 4. The induction of DNA damage and DNA repair signals during and after replication stress. A) RPA-CFP (magenta) and Rad52-YFP (green) foci in cells during experiment (bottom), compared to transmitted light image after 4h, 36°C. 10 µm scale bar. B-D) Focus formation in each genotype for repair (Rad52, B), ssDNA (RPA, C) and the co- localization of Rad52 with RPA (D). Proportions from pooled data of 2 or more independent experiments, and 95% CI. E) A de-magnified, tri-colour comparison of the whole nucleus with histone-RFP (top) is compared to 3D-SIM z-sections of a Rad52-YFP RPA-CFP megafocus. From a mcm4-degron culture after 4h 36°C. Comparative scale bars in each row (left-most section) are 1 µm. F) Pulsed field gel electrophoresis (PFGE) in asynchronous (AS) cells, after 4h 36°C (4h) and 2h release to 25°C (R). S. pombe chromosomes (left) and low molecular weight DNA (LMW) are indicated. Images taken from 2 gels run in parallel (line indicates image break). LMW DNA is a symptom of double strand breaks (DSBs (Froget et al, 2010)). The analysis of PFGE data (right), from 3 biological replicates, shows that DNA signal trapped in the well increases in mcm4- degron relative to the total signal per lane, and suggests increased replication intermediates in mcm4-degron. In contrast, LMW signal (right, DNA damage) increases in mcm4-ts, suggesting DSBs and/or sheared chromosomes. Figure S5, related to Figure 5. DNA damage, repair and checkpoint control in other replication mutant backgrounds A) RPA-CFP and Rad52-YFP foci in two established initiation mutants, orp1ts (orp1-4) and rad4ts (rad4-116). RPA-CFP is shown as a heat map on the left (as described in Fig 4A), and as a thresholded (non-heatmap) signal coloured magenta in the merged image. Rad52-YFP foci (green) are thresholded signals coloured green in both single channel and merged image. Scale bar is 10 µm. B,C) Proportions of cells with Rad52-YFP and RPA-CFP foci, are presented with 95% CI at timepoints as described in scheme (B, top). D) Cell length measured from cell tip to tip, or tip to septum, in over 100 cells and 2 biological replicates. E) The proportion of dead cells over time. Dead cells appear crinkled in transmitted light images (e.g. * in rad4ts, panel A). F) Chromosome mis-segregation events are plotted for orp1ts and rad4ts cells during the experiment, with 95% CIs. Figure S6, related to Figure 5. DNA damage, repair and checkpoint control through Chk1, Mus81. A) Representative cells of each genotype stained for DNA and septa, 10 µm scale bar, taken at 4h 36°C. B) Chk1 prevents some division (black bars), and all “cuts” (grey) in mcm4-degron at 36°C only, while loss of Chk1 allows division in mcm4-ts chk1∆. Cells, sampled according to the experiment outline (A) were stained to detect nuclei and septa. C) Loss of Mus81 endonuclease promotes division in mcm4-ts mus81∆ causing aneupoid and cut cells. Representative image of DNA/septum stained cells used in Fig 4F. Experiment outline shown below. D,E) The proportion of cells with DNA fragments (i.e. potential micronuclei) or unequal divisions (reductional anaphase, potential aneuploidy) does not increase in mcm4-degron mus81∆ during recovery at 25°C (R). However, mcm4-ts mus81∆ shows a significant increase in these phenotypes after 2h 25°C. Statistical significance values by 2-tailed z-test, comparing genotype in mus81+ versus mus81∆ at 2h release. Table S1. Strains from Forsburg yeast (FY) collection. Strain (FY) Genotype Figure 254 h- can1-1 leu1-32 ade6-M210 ura4-D18 4, S4, S6 421 h- ade6-704 leu1-32 ura4-D18 ∆chk1::ura4+ S6 784 h+ cdc21-M68 ura4-D18 leu1-32 ade6-M210 can1-1 4, S4, S6 1258 h+ his7+::lacI-GFP lys1+::lacO ade6-M210 leu1 ura4 S2 b 1963 h- cdc21-M68 chk1::ura4+ ura4-D18 S6 2317 h+ leu1-32::hENT1-leu1+(pJAH29) his7-366::hsv-tk- his7+(pJAH31) ura4-D18 ade6-M210 1A, 1D, S1, 3A, 3B, S3 2514 h+ leu1-32::hENT1-leu1+(pJAH29) his7-366::hsv-tk- his7+(pJAH31) cdc21-M68 leu1-32 ura4-D18 ade6-M210 1A, 1D, S1, 3A, 3B, S3 2518 h+ leu1-32::hENT1-leu1+(pJAH29) his7-366::hsv-tk- his7+(pJAH31) cdc19-P1 ura4-D18 ade6-M210 1A, 1D, S1 3288 h- mus81∆::KanMX ura4-D18 ade6-M210 4, S6 3395 h- mcm4(cdc21-M68)-ts-dg::ura4+ ura4-D18 4, S4, S6 3730 h- mus81∆::kanMX mcm4(cdc21-M68)-ts-dg::ura4+ ura4-D18 ade6- 4, S6 4611 h- chk1HA(ep) ade6-M216 ura4-D18 leu1-32 4D 4743 h- rad11-Cerulean::hphMX rad22-YFP-natMX leu1-32 ade6-M210 ura4-D18 4A, S4 4744 h+ rad11-Cerulean::hphMX rad22-YFP-natMX sad1-DsRed- LEU2+ leu1-32 ade6-M210 ura4-D18 can1-1 S1 4819 h? cdc21-M68 mus81∆::KanMX leu1-32 ura4-D18 can1? ade6- M216 4, S6 4855 h+ mcm4(cdc21-M68)-ts-dg::ura4+ sad1-DsRed-LEU2 rad11- Cerulean::hphMX rad22-YFP-natMX leu1-32 ura4-D18 S1 4856 h+ cdc21-M68 sad1-DsRed-LEU2 rad11-Cerulean::hphMX rad22-YFP-natMX leu1-32 ade6-M210 ura4-D18 S1 4857 h- cdc21-M68 rad11-Cerulean::hphMX rad22-YFP-natMX leu1- 32 ade6-M210 ura4-D18 4A, S4 5279 h- mcm4-(cdc21-M68)ts-dg::ura4+ ade6-M216 leu1-32 ura4-D18 rad22-YFP-natMX rad11-CFP-hphMX 4A, S4 6355 h- chk1HA(ep) cdc21-M68 ade6-M216 ura4-D18 leu1-32 can1-1 (mcm4) 4D 6497 h+ cdc21-ts-dg::ura4+ ura4-D18 chk1HA(ep) ade6-M216 leu1- 32 4D 6546 h- mcm4(cdc21-M68)-ts-dg::ura4+ rad11-CFP-hphMX rad22- YFP-NatMX hht1-mRFP:KanMX6 ura4-D18 leu1-32 2B, 4B, V1 a , V2, V4, V5, V6 6547 h- cdc21-M68 rad11-CFP-hphMX rad22-YFP-NatMX hht1- mRFP:KanMX6 ura4-D18 leu1-32 ade6-M210 2B, S4, V1, V2, V6 6583 h- rad11-CFP-hphMX rad22-YFP-NatMX hht1-mRFP:KanMX6 ura4-D18 leu1-32 ade6-M210 2B, S4, V1, V2, V6 6599 h- mcm4(cdc21-M68)-ts-dg::ura4+ ura4-D18 cnp1-mCherry- kanMX6 Rad52-YFP-NatMX rad11-CFP-HphMX leu1-32 ade6- M210 S2 6601 h- ura4-D18 cnp1-mCherry-kanMX6 Rad52-YFP-NatMX rad11- CFP-HphMX leu1-32 ade6-M210 S2 6604 h- cdc21-M68 ura4-D18 cnp1-mCherry-kanMX6 Rad52-YFP- NatMX rad11-CFP-HphMX leu1-32 ade6-M210 S2 6965 h+ cdc21-M68-ts-dg(mcm4-degron)::ura4+ ura4-D18 leu1- 32::[hENT leu1+] his7-366::[hsv-tk his7+] ade6-M216 1A, 1D, S1, 3A, 3B, S3 6980 h? mcm4(cdc21-M68)-ts-dg::ura4+ hht1-mRFP:kanMX his7+::lacI-GFP lys1+::lacO leu1-32 ura4-D18 S2 b 7395 h? hht1-RFP::KanMX6 leu1-32 arg3+::ccr1N-GFP::his5+ ura4- D18 his5D? control for 2A 7396 h? cdc21-M68-ts-dg::ura4+ hht1-RFP::KanMX6 arg3+::ccr1N- GFP::his5+ ura4-D18 his5D? 2A, S2, V3 7422 h+ mcm4(cdc21-M68)-ts-dg::ura4+ arg3+::ccr1N- mCherry::his5+ his5D? rad11-CFP-hphMX rad22-YFP-NatMX ura4-D18 3D, V6, V7 7426 h- arg3+::ccr1N-mCherry::his5+ his5D? rad11-CFP-hphMX rad22-YFP-NatMX ura4-D18 leu1-32 control for 3D a Supplemental Video 1 (V1); other Videos labeled similarly b Obtained from M. Yanagida; strain described in 1 c Obtained from S. Kearsey; strain described in (Lindner, 2002) d Tagged ccr1 N-terminal tagged proteins obtained from Xie Tang, Z. Cande lab. Described in the reference as as D817-GFP and D817-mCherry 2 Table S2. Primers used in qPCR reactions. Amplicon Stock number Sequence Reference 2004-F 1257 CGGATCCGTAATCCCAACAA 3 2004-R 1258 TTTGCTTACATTTTCGGGAACTTA 3002-F 1267 TCATTAGCAAACAAAAGCAATTGAG 3 3002-R 1268 AATTTCCGGGCATTAAAAACG CEN-F (dg) 1185 TATCCTGCGTCTCGGTATCC 4 CEN-R (dg) 1186 CTGTTCGTGAATGCTGAGAAA Video S1, Cell divisions at 36°C, related to Figure 1 Video microscopy of cells at 36°C (6h total time) monitors histone (magenta; hht1-RFP), ssDNA (green; RPA-CFP) and transmitted light image of cells. The division patterns change with genotype (FY6546, 6547, 6583) in each representative field. Video S2, histone/dsDNA during division at 25°C, related to Figure 1 Cells treated for 4h at 36°C were released to 25°C and monitored over 12h for histone (magenta; hht1-RFP), ssDNA (green; RPA-CFP) and transmitted light images. Strains (FY6546, 6547, 6583) were loaded in a microfluidics plate under constant flow; representative videos shown (minimum 2 biological, 6 experimental replicates for each strain). Video S3, structure of an apparent micronucleus, related to Figure 2 Rotational view of an mcm4-degron cell after 4h at 36°C showing membranes (green, ccr1N- GFP) and chromatin (magenta, hht1-RFP) during release (2h, 25°C). The image is constructed from z-sections, and DNA volume-surface rendered in Imaris. The micronucleus and primary nucleus show membrane projections that are not connected. Instead, the membrane connected to the micronucleus connects to the septum. Video S4, continued DNA mis-segregation and multiple spindle attachments in mcm4- degron divisions after replication stress, related to Figure 2 A micronucleus forms in the top left cell during the first division, is partitioned to one daughter in the next division, and then divides itself in a granddaughter cell. Cells (mcm4-degron after 4h 36°C) were imaged for histone (magenta) and membrane (green) signals over 16h at 25°C. Video S5, repeated segregation/fusion cycles of apparent micronuclei in mcm4-degron divisions after replication stress, related to Figure 2 A micronucleus is present and resorbs before cell division in mcm4-degron cells monitored for 16h at 25°C. Subsequent divisions also form micronuclei, which frequently rejoin the parent nucleus. Cells were first heat treated (4h, 36°C) and monitored for chromatin (magenta) and membrane (green) as in Video 7. Video S6, dynamic ultrafine DNA bridge coated with RPA, related to Figure 2 Dynamic ssDNA bridges (blue, RPA-CFP) and sporadic repair foci (yellow, Rad52-YFP) are formed in an mcm4-degron cell during release at 25°C (post-36°C, 4h). See also panels, Fig 2C. Video S7, 3D-structured illumination microscopy reconstruction of a megafocus, related to Figure 4 Volume rendering and surface reconstruction of 3D SIM image of mcm4-degron nuclei after 4h 36°C. The megafocus of ssDNA (blue, RPA-CFP) and Rad52 (yellow, Rad52-YFP) is located in chromatin-depleted pockets of the nucleus (magenta, hht1-RFP). Video S8, Rad52 repair foci persist into mitosis during mcm4-degron divisions after replication stress, related to Figure 5 Mutant cells divide with Rad52 repair foci during release (12h, 25°C) after 4h 36°C. Identical fields as Video S2, but shown with histone (magenta) and Rad52-YFP (green). Supplemental References 1 Nabeshima, K. et al. Dynamics of centromeres during metaphase-anaphase transition in fission yeast: Dis1 is implicated in force balance in metaphase bipolar spindle. Mol. Biol. Cell 9, 3211-3225 (1998). 2 Tang, X., Jin, Y. & Cande, W. Z. Bqt2p is essential for initiating telomere clustering upon pheromone sensing in fission yeast. J. Cell Biol. 173, 845-851, doi:10.1083/jcb.200602152 (2006). 3 Nitani, N., Yadani, C., Yabuuchi, H., Masukata, H. & Nakagawa, T. Mcm4 C-terminal domain of MCM helicase prevents excessive formation of single-stranded DNA at stalled replication forks. Proc. Natl. Acad. Sci. U S A 105, 12973-12978, doi:10.1073/pnas.0805307105 (2008). 4 Kloc, A., Zaratiegui, M., Nora, E. & Martienssen, R. RNA interference guides histone modification during the S phase of chromosomal replication. Curr. Biol. 18, 490-495, doi:10.1016/j.cub.2008.03.016 (2008). 61 Chapter 3 Characterization of a novel MMS-sensitive allele of S. pombe mcm4 + In this chapter I characterize a novel phenotype associated with a truncation allele of S. pombe Mcm4. This mutant which lacks 106 amino acids from the C terminus; was shown to be HU- sensitive and temperature-sensitive. (Nitani et al. 2008). We observed that in addition to these two phenotypes the mutant was also MMS-sensitive which has not been observed in Mcm4 alleles. The chapter describes the structure function analysis carried out on the Mcm4 c106 truncation and how it compares to the canonical mcm4-ts. 62 Characterization of a novel MMS-sensitive allele of S. pombe mcm4 + ABSTRACT: The minichromosome maintenance (MCM) complex is essential for the assembly and maintenance of a replication fork. The Mcm4 subunit in Schizosaccharomyces pombe is known to play a role in response to replication stress caused by hydroxyurea. In this work we further characterize a temperature sensitive C-terminal truncation allele of Schizosaccharomyces pombe mcm4 +. Uniquely amongst known mcm4 alleles, this mutation causes sensitivity to the alkylation damaging agent methyl methanesulfonate (MMS). The cells show increased repair foci of RPA and Rad52 even in the absence of treatment, and these increase in the presence of MMS. The mcm4-c106 mutant is synthetic lethal with mutations disrupting fork protection complex (FPC) proteins Swi1 and Swi3, and the Chk1 damage checkpoint . Surprisingly we found that the deletion of rif1 + suppressed the MMS sensitive phenotype. Together, these data suggest that mcm4-c106 destabilizes replisome structure. INTRODUCTION: The eukaryotic MCM helicase comprises six different, related subunits (Mcm2-7) each of which is essential for eukaryotic DNA replication (review in Forsburg, 2004, Bochman & Schwacha, 2009). All minichromosome maintenance (MCM) proteins share a distinct ATPase motif known as the “MCM Box” (reviewed in Forsburg, 2004, Bochman & Schwacha, 2009) that includes consensus Walker A and Walker B ATPase domains, and an arginine finger. The MCM complex is required for replication initiation and is associated with the replication fork throughout DNA 63 synthesis. Helicase activity in vivo depends upon all six MCMs as well as Cdc45 and GINS to create a larger complex called CMG (Moyer et al. 2006). Mutations in MCM genes are linked to genome instability in mammalian systems. The point mutation F345I (chaos3) located downstream of the Zn-finger motif of MCM4 in mouse is associated with mammary carcinoma (Shima et al. 2007). The mcm4-D573H mutation is associated with T cell lymphoblastic leukemia/lymphoma in mouse model (Bagley et al. 2012) and mcm4-G364R in humans is associated with skin cancer (Ishimi and Irie 2015). All these are associated with increased double strand breaks, and in some cases formation of micronuclei. N-terminally truncated Mcm4 (∆1-50 ) is linked to glucocorticoid deficiency, and defective DNA repair in humans (Hughes et al. 2012, Gineau et al. 2012). Although the primary sequence of Mcm4 N-terminus is neither conserved nor essential, this domain appears to be a common substrate for the DDK kinase required to initiate replication (Masai et al. 2006, Sheu and Stillman 2006). In budding yeast, deletion of the N-terminus bypasses a requirement for DDK, suggesting that DDK overcomes an inhibitory function (Sheu and Stillman 2010). The N- terminus is also important for regulating fork progression when cells have depleted nucleotide pools during hydroxyurea (HU) treatment (Devault et al.2008, Sheu et al. 2014). The fission yeast mcm4 + (cdc21 + ) gene was originally identified in a screen for temperature sensitive cdc mutants that arrest as elongated cells with undivided nuclei at the restrictive temperature (Nasmyth &Nurse, 1981, Coxon et al.1992). These mcm4-M68 cells accumulate approximately 2C DNA content and show evidence of DNA damage including DNA double strand breaks and generating a robust checkpoint-dependent arrest (Nasmyth & Nurse, 1981, Coxon et al. 1992, Liang et al. 1999, Bailis et al. 2008,Sabatinos et al. 2015). Viability is 64 low upon return to permissive temperature, suggesting this damage is irreversible (Liang et al. 1999, Bailis et al. 2008). The 2C DNA content observed in mcm4-M68 suggests that cells are competent for replication initiation and the bulk of DNA replication at the restrictive temperature. A second temperature sensitive allele was constructed by fusing a degron cassette to mcm4-M68 (Lindner et al. 2002). This enhances protein turnover leading to a rapid inactivation at restrictive temperature and cells arrest with a 1C DNA content (Lindner et al. 2002, Bailis et al. 2008, Sabatinos et al. 2015). However, despite evidence of DNA damage including large RPA and Rad52 containing “megafoci”, these cells continue to divide, indicating that they have evaded the damage checkpoint (Sabatinos et al. 2015). Survivors show dramatic evidence for chromosome mis-segregation, abnormal nuclear division, and chromosome rearrangement (Sabatinos et al. 2015). C-terminal truncations of Mcm4 cause HU sensitivity, and fail to restrain single-stranded DNA accumulation (Nitani et al. 2008). A large C-terminal truncation mutant mcm4-c106 is both temperature sensitive and HU sensitive (Nitani et al. 2008). In this study, we show that, unlike other mutant alleles of mcm4, mcm4-c106 is also sensitive to the alkylating agent MMS. Moreover, mcm4-c106 survives incubation at the restrictive temperature with high viability, in contrast to the other temperature sensitive alleles. Genetic interactions and synthetic lethality with components of the fork protection complex suggest that this mutation leads to specific defects in maintaining replisome structure. MATERIAL AND METHODS: Cell growth and cultures: Fission yeast strains are listed in Table 2. All strains were grown and maintained according to standard protocols (Sabatinos & Forsburg, 2010). Strains were grown in 65 YES or Edinburgh minimal medium (EMM) with ammonium chloride as the nitrogen source, supplemented with the required nutrients. Cells were grown in 25° unless otherwise stated. For all experiments cultures were grown in 5ml of liquid media from a single colony at 25° overnight and released to fresh media and grown at 25° to mid log phase. Serial dilutions and plating assays were performed in cultures grown in YES while the imaging experiments were performed in cultures grown in EMM. Serial dilution assays and relative viability: For serial dilutions, cell cultures were grown in 5ml of YES from a single colony at 25° overnight to mid log phase. Cells were counted and fivefold serial dilutions were prepared to ensure that equal numbers were plated on YES media containing the drug to assess drug sensitivity or grown at 25°, 32° and 36° to access the effects on temperature. Drug plates were allowed to grow for 3-5 days at 25° before scanning on a flatbed scanner. The experiments were repeated at least twice. For relative viability, cells at OD 595 ~0.3 were treated with 0.01% MMS for 4-6 hrs or shifted to 36° for 4-6hrs (as indicated in the figure legend). Samples were collected every 2 hrs and fixed in 70% ethanol for Flow Cytometry (FACS) and DNA staining with DAPI. At each time point cells were serially diluted 1:10, and equal volumes were plated at each time point and allowed to grow at 25° for 5 days before counting viable colonies. Protein extractions: Western blot analysis was performed using cultures grown to early log phase (OD595 ~0.3) in YES at 25°. Cultures were split into equal volumes and treated with 0.01% MMS or left 66 untreated for 4hrs at 25°. 10X stop buffer containing 2% Sodium Azide was added and cultures were incubated on ice for 10mins before harvesting the cells. Cells were subsequently washed twice with 1X PBS and whole cell proteins were extracted using trichloric acid (TCA) (Foiani et al. 1994). The extractions were quantified using Pierce BCA Kit. Equal amounts of 80-100ug of protein was loaded on 8% SDS PAGE gels. Primary antibody for Chk1HA (16B12 anti-HA ;Covance and anti-HA; Abcam) were used in 1:1500 dilutions overnight at 4°. Mcm4 protein levels were detected with antibody purified from rabbit serum 5898 (1:3000) (Sherman, et.al., 1998) incubated at 4° over night. After washing with PBST; anti-mouse-IgG-HRP secondary antibody (1:5000; sigma) was used to detect HA, while Mcm4 was detected using anti rabbit - HRP (1:5000; BD Biosciences) incubated for 1hour at room temperature. PCNA anti-mouse (1:1500; Santa Cruz) was used as the loading control. Pulse Field gel electrophoresis (PFGE): Pulse field gel electrophoresis to separate full-length chromosomes was performed upon 50ml cultures grown to early log phase (OD 595 ~0.3-0.4). Cultures were shifted to 36° for 4hrs and released to permissive temperature 25° for 2hrs. For the MMS treatment the cells were treated with 0.01% MMS for 4hrs and released to media lacking MMS for 2hrs for recovery after washing out the drug from the cultures. Cells were treated with 10X stop buffer containing 2% Sodium Azide and placed on ice for 5mins before harvesting the cells. Harvested cells were washed with 1XPBS and CSE buffer (20Mm Citric Acid, 20Mm Na 2 HPO 4 , 40Mm EDTA,1.2M sorbitol, at pH5.6 sterilized and stored at room temperature) Each culture was digested with 0.04 mg/ml 100T Zymolase and 0.45 mg/ml Sigma lysing enzymes in CSE Digested cells were used to prepare plugs which were resuspended in 1X TSE ( 10mM Tris pH 7.5, 45mM EDTA pH 8.0, 0.9M Sorbitol). Plugs were treated with proteinase K Sarkosyl/EDTA at 55° for 48hrs (1% 67 Sarkosyl, 0.5M EDTA pH 9.5). The buffer was changed after 24hrs of incubation and washed 4 times 30min each with 1XTE. Plugs were washed with 1X TAE prior to running the gels. Gel was run using a BioRad Chef II Pulse Field Machine for 48 hr using 2 V/cm, 1200-1800-sec switch time, and a 106° angle. DNA was visualized via Ethidium Bromide staining. Flow Cytometry ( FACS): Flow cytometry was performed as described in (Sabatinos & Forsburg, 2015). Briefly cells were fixed in 70% ice cold ethanol, washed with 50mM sodium citrate and re-suspended in 50mM sodium citrate with 0.1mg/ml RNAse. Samples were next stained with 1µM Sytox Green (Invitrogen) in 50mM sodium citrate and sonicated at 20% amplitude for 5 sec. Samples were analyzed by running it on a Becton Dickinson FACScan flow cytometer. Microscopy: Cultures were grown in EMM supplement with ammonium chloride. Agar pads were prepared as described in (Green et al.2015). Live cells images were acquired with a DeltaVision Core (Applied Precision, Issaquah, WA) microscope using a 60x N.A. 1.4 PlanApo objective lens and a 12-bit Photometrics CoolSnap HQII CCD. The system x-y pixel size is 0.109µm. SoftWoRx v4.1 (Applied Precision, Issaquah, WA) software was used at acquisition. The image acquisition consisted of 13 Z-stacks with 0.5µm for visualizing Rad11 and Rad52 foci at 36° and MMS. Cells were visualized at the asynchronous stage, 4hrs post treatment and 2 hrs post release from the treatment. Movies were captured to look at the replication dynamics in real time. 18 Z-stacks with 0.5µm were acquired 10mins apart for the length of the experiment. The temperature was controlled at 25° if not specified. For still imaging CFP was excited and detected with an (ex)438/24, (em)470/24 filter set and a 0.5-sec exposure excitation intensity attenuated to 10%); 68 and YFP was excited and detected with an (ex)513/17, (em)559/38 filter set and a 0.5-sec exposure excitation intensity attenuated to 32%. Suitable polychroic mirrors were used. Ten 0.5µm serial z-sections were captured. 3-D stacks were deconvolved with manufacturer provided OTFs using a constrained iterative algorithm, images were maximum intensity projected for presentation. Images were contrast adjusted using a histogram stretch with an equivalent scale and gamma for comparability. Strains are available upon request RESULTS: A MMS sensitive allele of mcm4 + Mutants with defects in replisome components often show sensitivity to DNA damaging agents, but not all mutants are sensitive to all drugs. For example, in a recent study, we showed that cells deleted for non-essential helicases have distinct patterns of genotoxin sensitivity that establish a fingerprint for their roles in DNA replication and repair (Ding and Forsburg 2014). Here, we analyzed a panel of mutants with different mutations in the essential mcm4 + gene for their sensitivity to different damaging agents including (Nasmyth and Nurse 1981) HU, which depletes nucleotide pools and causes fork stalling (Thelander and Reichard 1979); MMS, an alkylating agent that generates diverse lesions that block DNA polymerase (Lundin et al. 2005); and camptothecin (CPT), a topoisomerase inhibitor that leads to S-phase specific double strand breaks (Liu et al. 2000). We examined the known temperature sensitive alleles mcm4-M68, , mcm4 ts dg and mcm4-c106 (Nasmyth and Nurse 1981, Lindner et al. 2002,Nitani et al. 2008). The 69 remaining mutants we tested include a deletion of the N-terminal residues 2-73, a single point mutation F346I corresponding to the chaos allele in mouse (J-P Yuan and SLF unpublished; (Shima et al. 2007)), and mcm4-4SA, which contains mutations in putative damage-specific phosphorylation sites S30A S38A S81A T95A (Figure 3.1A). As observed previously, some mcm4 mutants show sensitivity to HU including the temperature sensitive degron allele mcm4-dg, (Figure 3.S1A) and the C-terminal truncation alleles mcm4-c84 and mcm4-c106 (Nitani et al.2008). We didn’t observe CPT sensitivity in any of the above mutants (Figure 3.S1B). Unexpectedly, we observed that the temperature sensitive mcm4-c106 truncation is also sensitive to MMS exposure, which is not seen for any other mcm4 alleles (Figure 3.1B). The ts and MMS sensitive phenotypes were not observed for the mcm4-c84 truncation (Nitani et al.2008). These results indicate that the C terminus of Mcm4 is necessary for proper response to MMS. In general, sensitivity to MMS is associated with mutations that affect checkpoint response or repair, and mutations that disrupt a distinct subset of replisome components. These include mutations affecting the fork protection complex (FPC) proteins Swi1 and Swi3, and the MCM kinase Hsk1/DDK that interacts with FPC (Noguchi et al. 2003,Noguchi et al.2004, Sommariva et al. 2005, Dolan et al. 2010, Memisoglu & Samson, 2000,Kumar & Huberman, 2004). Given that mcm4-c106 shows sensitivity to MMS as well as to higher temperatures, we investigated both these phenotypes. mcm4-c106 cells have a unique replication phenotype There is no obvious difference in growth rate between wild type and mcm4-c106 cells at permissive temperature (data not shown). To assess whether there is a subtle 70 replication defect, we performed a classic minichromosome maintenance assay (Tye 1999) to examine the transformation efficiency of an origin-containing plasmid at permissive temperature. We transformed the wild type, mcm4-M68, and mcm4-c106 strains with a plasmid (pUR19N;(Barbet et al. 1992)) containing a single copy of S. pombe ars1 and compared the number of transformants/µg DNA (transformation efficiency) and plasmid stability (colony size). We found that both the number of transformed cells and the size of the colonies were reduced in mcm4-c106 compared to either mcm4-M68 or wild type at permissive temperature (Figure 3.2 A, B). Next, we examined a plasmid with an additional ars (pDblet), which is intrinsically more stable (Brun et al. 1995). We observed that transformation efficiency of mcm4-c106 improved, and transformant colonies were larger than when transformed with a single ars plasmid. (Figure 3.2C,D). This partial rescue by an additional ars element suggests that this mutant allele has a defect in replication initiation at permissive temperature. At 36°, mcm4-M68 loses viability rapidly (Liang and Forsburg 2001). We examined the relative viability of mcm4-c106 following a shift to the restrictive temperature, but the loss of viability was more modest compared to mcm4-M68 (Figure 3.3A). We examined DNA accumulation using flow cytometry on cells that were arrested in G1 by nitrogen starvation, and released to the permissive temperature (25°), and the restrictive temperature (36°) (Figure 3.3B). We observed DNA accumulation to approximately 2C DNA content in both wild type and mcm4-c106 cells even at restrictive (36°) and non restrictive (25°) temperatures. This is similar to observations for the original mcm4-M68 temperature allele, which has a late S phase arrest (Nasmyth & Nurse, 1981, Coxon et al. 1992, Bailis et al. 2008, Sabatinos et al. 2015). 71 However, the chromosome profiles observed in pulsed field gel electrophoresis (PFGE) were strikingly different between these two mcm4 alleles. Typically, the chromosomes from cells with replication defects do not migrate normally at restrictive temperature, either due to unresolved replication or recombination intermediates that preclude migration, or due to chromosome breakage (e.g., (Liang and Forsburg 2001, Waseem et al. 1992)). Surprisingly, mcm4-c106 showed intact chromosomes under all conditions (Figure 3.3C), while as seen previously, the mcm4-M68 chromosomes do not migrate at their normal position during a 36° temperature shift or upon release to 25°, but are replaced by a smear. This is consistent with unresolved replication intermediates and also the double strand breaks reported earlier (Liang & Forsburg, 2001, Bailis et al. 2008, Sabatinos et al. 2015 ). This, along with the maintenance of viability and ability to recover from temperature arrest, suggests that the nature of the defect in mcm4-c106 is different from that of the well-studied mcm4-M68. We observed a modest loss of viability of mcm4-c106 cells treated with MMS (Figure 3.3D), consistent with the MMS sensitivity observed in plate assays (Figure 3.1B). During MMS treatment in liquid culture at the permissive temperature both wild type and mcm4-c106 mutants showed an S phase delay as indicated by the intermediate peak that is observed in the FACS profiles (Figure 3.3B). The loss of viability was relatively modest compared to a repair-defective allele of PCNA, pcn1-K164R (Frampton et al. 2006). In both wild type and mcm4-c106 cells treated with MMS and following release, we observed little if any migration of the chromosomes into the gel (data not shown). Finally, we examined Mcm4 protein levels in the mutant. Loss of Mcm4 protein has been correlated with genomic instability (Liang & Forsburg, 2001, 72 Bailis et al. 2008, Sabatinos et al. 2015). However, we saw no change in Mcm4 protein levels during MMS treatment or at 36° in mcm4-c106 (Figure 3.4C, D),suggesting its temperature sensitivity and MMS phenotypes are not related to protein levels. Chromosome segregation is normal in mcm4-c106 Recently, we showed that mcm4-ts-dg mutants undergo division despite their replication defects, and this is accompanied by aberrant nuclear division, abnormal chromosome segregation and reduced viability (Sabatinos et al. 2015). We saw no evidence for abnormal mitosis in mcm4-c106 cells at permissive temperature. To examine this more closely, we determined segregation of chromosome I, using a lacI- GFP fusion in a strain with a lacO array at centromere I to generate a centromere proximal signal (Nabeshima et al. 1998). We observed no evidence for lagging chromosomes, or chromosome mis-segregation , indicating no substantial mitotic defects in mcm4-c106 (Figure 3.S2). mcm4-c106 requires an intact damage checkpoint The mcm4-c106 cells elongate following treatment at 36° or in MMS which suggests successful activation of the damage checkpoint. We verified this by monitoring the checkpoint kinase Chk1, which undergoes an activating phosphorylation that results in a mobility shift in SDS-PAGE (Walworth and Bernards 1996). We observed a shift in Chk1 in both wild type and mcm4-c106 cells treated with MMS (Figure 3.4A), and in wild type and mcm4-c106 cells at the restrictive temperature (Figure 3.4B), consistent with successful activation of Chk1 under both conditions in the mutant. However, we 73 observed no evident Chk1 phosphorylation in asynchronously growing cultures at 25° in the absence of treatment (Figure 3.4A,B). Despite the lack of a Chk1 phosphorylation shift at permissive temperatures, we found that double mutants between the mcm4-c106 and either rad3∆ or chk1∆ were inviable. However double mutants with the S phase checkpoint mutant cds1∆ were viable (Table 3.1). We conclude that even though we do not observe shifted mobility of Chk1 at permissive temperature, there is sufficient damage even in unperturbed cells to cause them to depend upon this checkpoint for viability. Repair foci accumulate in mcm4-c106 Previously, we examined replication stress by examining the accumulation of repair foci corresponding to the single strand DNA binding protein RPA (labeled with CFP), or the recombination protein Rad52 (labeled with YFP), and have observed differences in their distribution, pattern, and intensity in different conditions (Bailis et al. 2008, Sabatinos et al. 2012,Sabatinos et al. 2015 ). There are dramatically different phenotypes between two different temperature sensitive alleles: mcm4-M68 forms multiple small foci and robustly arrests division at permissive temperature, while mcm4- dg forms a single large mega-focus, and fails to block division (Bailis et al. 2008, Sabatinos et al. 2015). We therefore examined repair foci fluorescence in both wild type and mcm4-c106 cells either shifted or released from 36°, or from MMS (Figure 3.5). At 25°, mcm4-c106 shows a modest increase in cells with both foci compared to wild type, consistent with an increased basal level of stress (Figure 3.5A,B,C). Following a shift to 36°, mcm4-c106 accumulated numerous small foci of RPA-CFP and Rad52- 74 YFP and these remain after 2 hours of release, whereas the foci in wild type cells decline by 4 hours at 36° (Figure 3.5A,B,C) (Sabatinos et al. 2015). The mcm4-c106 cells did not divide in the first two hours following release. This suggests multiple dispersed damage sites, similar to mcm4-M68. In wild type cells during a 4 hour treatment with 0.01% MMS or at 2 hours after release from MMS, there is modest increase in cells with RPA or Rad52 foci. This increase is measured from approximately 20% in untreated cells to about 50% in treated cells, but most of these have just one or two foci. In contrast, while mcm4-c106 cells have similar overall levels of focus formation in untreated cells, up to 80% of the cells have at least one focus in treated cells, and a strikingly large fraction contains multiple bright signals, which persist through the period of release. The majority of the RPA foci observed overlap with Rad52 foci (Figure 3.5A,C,D). Therefore, there is evidence for constitutive repair foci in mcm4-c106 cells which are dramatically increased upon exposure to MMS. rif1∆ rescues mcm4-c106 MMS phenotype One response to replication stress is to activate dormant origins (reviewed Alver et al. 2014). Previous work showed that mcm6-S1 mutant, which affects another subunit of the MCM complex, also displays MMS sensitivity (Maki et al. 2011). Deletion of the S-phase cyclin cig2 rescues this sensitivity, presumably by delaying G1/S phase and allowing additional licensing of origins (Maki et al. 2011). Therefore, we examined a double mutant of cig2∆ mcm4-c106. In contrast to the results reported for mcm6-S1, we observed only a very slight suppression of MMS sensitivity (Supplementary Figure 3.2). 75 In contrast, a double mutant rif1∆ mcm4-c106 showed a dramatic rescue of MMS sensitivity (Figure 3.6B). Rif1 has recently been identified as an antagonist of DDK kinase-mediated phosphorylation of MCM, and regulates timing of origin firing (Hayano et al. 2012,Yamazaki et al. 2013, Davé et al. 2014, Mattarocci et al. 2014, Hiraga et al. 2014). Despite this dramatic rescue of the MMS phenotype, however, rif1∆ does not rescue the temperature sensitivity of mcm4-c106 (Figure 3.6A). Mcm4-c106 requires fork protection complex for viability There is a central replisome scaffold that links the leading and lagging strand polymerases and the MCM helicase (Noguchi et al. 2004, Lou et al. 2008, Tanaka et al. 2009, Sommariva et al. 2005,Gambus et al. 2009). This scaffold consists of Mcl1/ScCtf4, Mrc1, and the fork protection complex (FPC) Swi1/ScTof1 and Swi3/ScCsm3 (reviewed in Aze et al. 2013). Mutants defective in these non-essential proteins are all sensitive to MMS, indicating that robust coupling of the helicase and polymerase is required for response to alkylation stress. We observed that similar to mcm4-c106, the MMS sensitivity associated with swi1∆ and swi3∆ is suppressed in rif1∆ double mutants (Figure 3.6C; Table 3.1), suggesting a related function. Therefore we tested epistasis between mcm4-c106 and FPC components. Unexpectedly, we observed that double mutants between mcm4-c106 and swi1∆, swi3∆, or mrc1∆ are all inviable even at 25°. Deletion of rif1∆ did not rescue the inviability of swi1∆ mcm4-c106 or swi3∆ mcm4-c106 strains. A double mutant between mcl1-1 temperature sensitive strain (Sc CTF4); (Williams and McIntosh 2002) and mcm4-c106 was viable but grew so poorly, it was impossible to assess its MMS sensitivity (Table 3.1). 76 The DDK kinase Hsk1 (ScCdc7) is essential for DNA replication, in part due to its phosphorylation of MCM proteins (Sheu & Stillman, 2006, Masai et al. 2000, Masai et al. 2006). It also interacts with the fork protection complex (Sommariva et al. 2005, Masai et al. 2006) and antagonizes Rif1( Hayano et al. 2012, Davé et al. 2014). Previously, we showed that the temperature sensitive mutant hsk1-1312 is sensitive to MMS and that Hsk1/DDK persists on the chromatin during MMS treatment, dependent upon the C-terminus of the regulatory subunit Dfp1 that is disrupted in the dfp1-r35 mutant allele (Dolan et al. 2010). We observed that the double mutants of mcm4-c106 hsk1 1312 and mcm4-c106 dfp-r35 formed microcolonies that could not be propagated. The Ctf18 protein is part of an alternative replication factor C complex RFC clamp loader (Mayer et al. 2001,Hanna et al.2001) that is associated with DNA polε (García-Rodríguez et al. 2015). In budding yeast and humans, Ctf18 associates with two additional subunits, Dcc1 and Ctf8, to form a heptameric complex that has been shown to have a role in sister chromatid cohesion (Mayer et al. 2001,Gellon et al. 2011). Additionally, ctf18 is lethal with swi1∆ and swi3∆ (Ansbach et al. 2008). We find that double mutants mcm4-c106 ctf18∆ and mcm4-c106 ctf8∆ are viable, but with a reduced permissive temperature (32°) (Figure 3.7A). However, they show no change in MMS sensitivity relative to their parents (Figure 3.7B; Table 1). Chl1 is a helicase linked to the lagging strand that is a high copy suppressor of swi1∆ damage sensitivity (Ansbach et al. 2008). The deletion chl1∆ is lethal when combined with ctf18∆ (Ansbach et al. 2008). Double mutants with mcm4-c106 and chl1∆ show increased sensitivity to MMS compared to their parent, but no effects on temperature (Figure 3.7C). 77 Swi1 and Swi3, the Ctf`18 complex, and Hsk1 have all been linked to defects in chromosome cohesion ( Bailis et al. 2003, Ansbach et al. 2008, Rapp et al. 2010). A temperature sensitive mutation affecting the cohesion subunit rad21-K1 combined with ctf18∆ shows increased sensitivity to MMS compared to the parents (Ansbach et al. 2008). Therefore we examined a double mutant mcm4-c106 rad21-K1. This strain is viable and shows a similar MMS sensitivity to parent rad21-K1 (Figure 3.7D). We tested several other mutations affecting proteins associated with the core replisome. Both temperature sensitive mutations cdc20-M10 and pol1-1, respectively encoding the leading strand DNA polymerase ε (D’Urso and Nurse 1997), and polymerase α (D’Urso et al. 1995) were viable in combination with mcm4-c106, while a temperature sensitive allele cdc6-23 affecting the lagging strand DNA polymerase δ (Iino and Yamamoto 1997) is lethal (data not shown). Mcm4-c106 interactions with repair pathways Finally we examined genetic interactions with other mutants in the MMS response pathway. The post replication repair (PRR) pathway includes both error free and error prone branches that facilitate the bypass of base lesions (reviewed in Huang & D’Andrea, 2006, Ulrich & Walden, 2010). These are regulated by levels of ubiquitylation on PCNA (Frampton et al. 2006). We examined double mutants of mcm4-c106 with repair mutants mms2∆, ubc13∆, pcn1-K164R and rad8∆. The double mutants were significantly more MMS sensitive than either single mutant. None of these mutants showed a growth defect in the absence of MMS (Figure 3.S4A). No genetic interactions 78 were observed in double mutants affecting the error prone polymerases rev3∆, rev1∆,polκ ∆,or eso1-∆ eta (Figure 3.S4B). Effects of mcm4-c106 in recombination defective mutants Replication fork stability and fork restart depend on proteins associated with recombination (reviewed in Lambert & Carr, 2013, Neelsen & Lopes, 2015). We constructed double mutants between mcm4-c106 and mutations that disrupt recombinational repair of damaged forks including: mus81∆ (endonuclease; (Boddy et al. 2001)), rad50 ∆ (MRN complex; (Bressan et al. 1999) rad51∆ (homologous recombination regulator; (Muris et al. 1993)), rqh1∆ (RecQ helicase; (Stewart et al. 1997) and srs2∆ (Wang et al. 2001, Maftahi et al. 2002). We find that rad50∆ mcm4- c106 is synthetic lethal, indicating that the constitutive damage of mcm4-c106 depends upon an active MRN complex. The mus81∆ double mutant had an extreme growth defect even at 25°, with slow growth and elongated cell morphology. In contrast, rad51∆,,rqh1∆, or srs2∆ double mutants were sensitive to MMS showing a sensitivity similar or greater than the most sensitive parent; but showed no growth defects were observed at permissive temperature (Figure 3.S5A,B,C).These data suggest that these proteins function in a pathway separate from the C-terminus of Mcm4. Discussion: Fission yeast Mcm4 is an essential subunit of the MCM helicase that is a critical component in the response to replication stress. Previous studies have shown that the Mcm4 carboxy terminal domain (CTD) is important for efficient recovery of HU-stalled replication forks, and a C-terminal truncation mcm4-c84 causes excessive formation of ssDNA when 79 replication is inhibited by hydroxyurea (Nitani et al. 2008). Nitani (Nitani et al. 2008) also identified a larger CTD truncation mutant mcm4-c106 as HU-sensitive, but due to its temperature sensitivity didn’t characterize it further. Our initial examination of the temperature sensitive phenotype of mcm4-c106 shows a distinct phenotype compared to other alleles of mcm4 + . Similar to the original mcm4-M68 strain (Bailis et al. 2008, Sabatinos et al. 2015), we observe that mcm4-c106 cells accumulate a 2C DNA content at restrictive temperature indicating substantial bulk DNA synthesis (Figure 3.3B). Cells elongate and do not divide, demonstrating successful activation of the checkpoint, consistent with a gel mobility shift of the Chk1 kinase (Figure 3.4B). The cells also show increased RPA and Rad52 foci during arrest and release, with small punctate morphology similar to that observed in mcm4-M68 (Figure 3.5A;Sabatinos et al. 2015). Strikingly, however, mcm4-c106 cells are able to recover with high viabilitiy from the temperature shift, which may be related to the observation that the chromosomes enter a pulsed- field gel normally both at restrictive temperature and following release, without the chromosome breaks and / or structural intermediates that impair chromosome migration in mcm4-M68. The temperature sensitive phenotype may reflect either Mcm4 protein unfolding (although the protein remains detectable) or some intrinsically temperature sensitive activity involved in replisome coupling that renders the C-terminus essential at high temperature. In any case, some fraction of the cells are competent to restart the cell cycle, indicating that the damage they suffer is not irreversible and that the MCM complex remains largely intact and located correctly in the nucleus, which is not seen for mcm4-M68 (Pasion and Forsburg 1999). Interestingly, we found that mcm4-c106 is also sensitive to alkylation damage caused by MMS treatment at the permissive temperature. The MMS sensitivity was not observed for other mcm4 alleles (Figure 3.1B). Previously, the only MMS-sensitive 80 MCM identified is an allele of mcm6 (mcm6-S1) defective in pre-RC assembly (Maki et al. 2011). MMS sensitivity is observed in mutants affecting a subset of other replisome components including Fork Protection Complex swi1∆, swi3∆, scaffolding protein mcl1, and the DDK kinase subunits hsk1-1312 or dfp1-r35 (Fung et al. 2002, Williams & McIntosh, 2002 , Sommariva et al. 2005; Dolan et al. 2010). FPC, its associated protein Mrc1, and Mcl1 (ScCtf4/AND-1) are associated with coupling of the helicase to the leading and lagging strands (Katou et al. 2003, Noguchi et al. 2004, Lou et al. 2008, Gambus et al. 2009). MMS treatment in fission yeast results in slowing of the replication fork (Chahwan et al. 2003, Kumar and Huberman 2004, Willis and Rhind, 2009). Although there have been reports that MMS generates DNA double strand breaks (Wyatt and Pittman 2006), this breakage may actually be an artifact of the procedure used in extracting DNA (Lundin et al.2005). A major form of recovery is template switching and re-priming (rev. Branzei and Foiani 2010), which leads to accumulation of single strand DNA and increased recombination intermediates (Willis and Rhind 2009, Koulintchenko et al. 2012). Formation of these recombination structures is disrupted in swi1∆ and swi3∆ , and also in rad2∆ mutants lacking the FEN1 flap endonuclease ( Noguchi et al. 2004, Koulintchenko et al. 2012).In budding yeast, DNA polymerase alpha, the Mcl1 orthologue Ctf4, and cohesin are also required for template switching, and evidence indicates this requirement is not limited to lagging strand lesions (Fumasoni et al. 2015). Given that these mutants displaying MMS sensitivity are all central components of the replisome, we predict that mcm4-c106 has a attenuated replisome structure even at 25° that impacts fork stability or template switching during MMS treatment Consistent with 81 this, we find that in minichromosome maintenance assays, mcm4-c106 shows a substantial reduction in plasmid transformation efficiency at the permissive temperature, even though it has no obvious growth defects. Since S. pombe plasmids do not have centromere-mediated segregation, efficiency of transformation is a metric for replication efficiency (Clyne and Kelly 1997). This result is consistent with a defect in initiation. There is a modest increase in the fraction of cells with repair foci at 25°. Although we see no evidence for Chk1 phosphorylation in unperturbed cells, a double mutant mcm4-c106 chk1∆ or mcm4-c106 rad3∆ is synthetically lethal, indicating the cells suffer sufficient damage to require an intact damage checkpoint. Consistent with this, we observe synthetic lethality with MRN component rad50∆, and severe synthetic sickness with mus81∆, which implicates fork processing and restart in the recovery from innate stress. in contrast, mcm4-c106 cells showed increased sensitivity to MMS is combination with mutations that directly affect downstream repair including homologous recombination, error-prone and error-free post-replication repair pathways. This would be expected if its defect is not in repair per se but in fork stability or recovery. We found that rif1∆ rescues the MMS sensitivity of mcm4-c106, but not its temperature sensitivity. We find that rif1∆ also rescues the MMS sensitivity of swi1∆ or swi3∆. Rif1 in budding yeast has a role in replication timing, via the recruitment of Glc7 phosphatase (Hayano et al. 2012, Davé et al. 2014, Mattarocci et al. 2014, Hiraga et al. 2014, Peace et al.2014). This antagonizes DDK mediated phosphorylation of Mcm4 that activates replication. Intriguingly, Rif1 is also proposed to modify the response to ssDNA that activates the checkpoint (Xu et al. 2010) as well as contributing to resection in break repair (Martina et al. 2014). In fission yeast, rif1∆ is reported to rescue hsk1-89 (Hayano 82 et al. 2012, Davé et al. 2014) and we find that it also rescues hsk1-1312 (J.P. Yuan and SLF, unpublished) . However Rif1 is not essential for cellular responses to replication stress (Hayano et al. 2012, Peace et al. 2014) . This rescue could occur either by moderating the checkpoint in some way, modulating ssDNA checkpoint dynamics or by activating otherwise dormant origins. Origin activation has been proposed to rescue the MMS sensitivity of mcm6-S1, another MCM subunit (Maki et al. 2011). However, unlike mcm6-S1, the MMS sensitivity of mcm4-c106 was not notably rescued by deletion of the S phase cyclin cig2. Interestingly, we observed synthetic lethality between mcm4-c106 and FPC mutants swi1∆, swi3∆, mrc1∆, and DDK mutants hsk1-1312 and dfp1-r35. Because we see no phenotype in double mutants with the replication checkpoint kinase cds1∆, we conclude that this synthetic lethality is not related to the S phase checkpoint activation by FPC (Noguchi et al. 2003) but rather, to the FPC’s structural role linking the helicase to the leading strand (Noguchi et al. 2004, Lou et al. 2008, reviewed Aze et al. 2013). Double mutants between mcm4-c106 and mcl1-1 temperature sensitive allele were viable, although significantly sicker than either parent. Mcl1/Ctf4 links the helicase to the lagging strand and DNA polymerase alpha through a direct interaction with the GINS subcomplex (Gambus et al. 2009, Simon et al. 2014). At least one arm of the helicase coupling system is required for viability, because mcl1-1 swi1 ∆ double mutants are synthetically lethal (J. P. Yuan and SLF, unpublished results). Therefore, one possibility is that mutation of mcm4-c106 may lead to defects in coupling to the lagging strand side 83 of the replisome, making it dependent upon the FPC and the leading strand coupling for viability even at permissive temperature. FPC, Ctf18, Mcl1 (Ctf4) and DDK are also associated with sister chromatin cohesion in S.pombe and other systems ( Hanna et al. 2001, Williams and McIntosh 2002, Bailis et al. 2003, Ansbach et al. 2008, Rapp et al. 2010). However, we observed no evidence for chromosome segregation errors in mcm4-c106 that would indicate a cohesion defect. A temperature sensitive mutation affecting the cohesion subunit rad21- K1 is synthetic lethal with either swi1∆ (Ansbach et al. 2008, Dolan et al. 2010, or hsk1- 1312 (Snaith et al. 2000), suggesting that leading strand coupling is essential when cohesion is attenuated. Intriguingly, studies suggest that cohesin may influence replication origin activity by affecting 3-D genome organization (Guillou et al. 2010,Yun et al. 2016). Mutations in rad21 are MMS sensitive, and lie in an epistatic pathway with rad50 + (Hartsuiker et al. 2001). Recently, cohesion has been shown to be required for efficient template switching in budding yeast in a pathway that includes Ctf4 (Sp Mcl1) (Fumasoni et al. 2015). Mcm4 has also been identified as a binding partner of mammalian Rad21 in two separate proteomics studies ( Guillou et al. 2010, Panigrahi et al. 2012). We observed reduced permissive temperature in ctf118∆ mcm4-c106, but no growth defects in rad21-K1 mcm4-c106. Additionally, the MMS sensitivity of rad21 mcm4-c106 is similar to that of the rad21-K1 parent. We propose that the C-terminus of Mcm4 potentially affects the lagging strand pathway, possibly via recruitment of cohesion to facilitate fork repair. 84 Together, these results indicate that mcm4-c106 has a novel replication defect, likely to do with replisome uncoupling that is distinct from that in other mcm4 conditional alleles. Along with our previous study (Sabatinos et al. 2015), this suggests that physiological inspection of conditional mutant phenotypes are likely to identify new domains and interactions that assemble and maintain the replicative helicase. ACKNOWLEDGEMENTS: We thank JiPing Yuan for help with strain construction, and Sarah Sabatinos for technical help and advice on the project. We thank Oscar Aparicio, and members of the Forsburg lab for helpful comments on the manuscript. This work was supported by NIH R01 GM081418, R01 GM111040, and R35-GM118109 REFERENCES: Alver, R. C., G. S. Chadha, and J. J. 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Kim et al., 2016 Reduced cohesin destabilizes high-level gene amplification by disrupting pre-replication complex bindings in human cancers with chromosomal instability. Nucleic Acids Res. 44: 558–72. Table 3.1: Sensitivity to MMS of double mutants of genes involved in different aspects of the cell cycle. Double mutant phenotypes between mcm4-c106 and other mutants in the indicated classes. Phenotype (a): viable means no change in temperature sensitivity. Synthetic lethal is dead at all temperatures. Sick shows reduced growth rate at all temperatures. <temp has reduced maximum growth temperature. MMS phenotype (b) is determined relative to the most sensitive parent (indicated) Category Mutant Function Phenotype with c106 a MMS Phenotype b Checkpoint cds1∆ Kinase; S phase/replication checkpoint Viable >mcm4c106 Checkpoint chk1∆ Kinase; G2/damage Synthetic Lethal ND 96 checkpoint Checkpoint mad2∆ Spindle checkpoint Viable =mcm4-c106 Checkpoint rad26∆ Checkpoint protein Synthetic Lethal ND Checkpoint rad3∆ Kinase Synthetic Lethal ND Cohesion chl1∆ Helicase Viable > mcm4c106 Cohesion rad21-K1 Cohesin protein Viable = rad21-K1 replication- FPC mcl1-1 ts Part of the FPC/cohesion Sick replication- FPC mrc1∆ Replication mutants/FPC Synthetic Lethal ND replication- FPC swi1∆ Replication mutants/FPC Synthetic Lethal ND replication- FPC swi3∆ Replication mutants/FPC Synthetic Lethal ND replication hsk1-1312 DDK kinase Synthetic Lethal ND replication dfp1-r35 DDK kinase Viable/sick ND replication, genome stability rif1∆ Rif1 antagonist of DDK Viable rescued genome brc1∆ Genome stability Viable >mcm4c106 97 stability replication- clamp loader ctf8∆ Cohesion- specific clamp- loader Viable < temp = mcm4c106 replication- clamp loader ctf18∆ Cohesion- specific clamp- loader Viable < temp = mcm4c106 genome stability fml1∆ Helicase; genome stability Viable =fml1 genome stability mus81∆ Holiday junction resolvase Sick =mus81 genome stability rqh1∆ Helicase; recombination antagonist Viable > mcm4c106 genome stability srs2∆ Helicase; recombination regulator Viable >mcm4c106 Repair eso1-∆eta Eso1- Polη fusion; deletes polymerase domain. error prone repair Viable = mcm4c106 Repair exo1∆ Exonuclease I Viable = exo1 Repair mms2∆ Ubiquitin ligase; error free repair Viable > mms2 98 Repair pcn1-K164R PCNA; ubiquitin site mutant Viable = pcn1-K164R Repair polk∆ Polκ ; error prone repair Viable >mcm4c106 Repair rhp51∆ Homologous recombination Viable =rhp51∆ Repair rad8∆ Ubiquitin ligase- helicase; error free repair Viable >rad8 Repair rev1∆ Deoxycytidyl transferase; error prone repair Viable ≥ mcm4c106 Repair rev3∆ Polζ Error prone repair Viable ≤mcm4c106 Repair rhp18∆ PCNA ubiquitin ligase Viable > rhp18 Repair ubc13∆ Ubiquitin ligase; error free Viable > ubc13 Other chp1∆ Heterochromatin protein Viable =mcm4c106 Other Cig2∆Cyc17 S phase cyclin Viable ≤ mcm4c106 Other reb1∆ Transcription termination Viable < temp =mcm4c106 99 Other swi6∆ Heterochromatin protein Viable = mcm4c106 Rad22∆ Viable /slow growing ND cdc20- M10(polε) Viable ND cdc6- 23(polδ) Synthetic lethal NA Psf2ts Component of the GINS replication complex Viable >mcm4c106 pol1-1(polα) Viable ND Rad50 ∆ Synthetic lethal NA 100 Table 3.2 : Strains used in this study: Strain Genotype Source FY 7 h- 972 Our stock FY 527 h- his3-D1 ade6-M216 ura4-D18 leu1-32 Our stock FY 528 h+ his3-D1 ade6-M210 ura4-D18 leu1-32 Our stock FY 261 h+ can1-1 leu1-32 ade6-M216 ura4-D18 Our stock FY 784 h+ cdc21-M68 ura4-D18 leu1-32 ade6-M210 can1-1 (mcm4) Our stock FY 4240 h- cdc21-c84:kan Takuro Nakagawa FY 4311 h- cdc21-c106::kan ura4-D18 his3-D1 ade6- M210 Our stock FY 4310 h- cdc21-c84::kan Ura4-D18 his3-D1 Our stock FY 5942 h- cdc21-c106::HphMx ura4-D18 his3-D1 ade6- M210 This work FY 3395 h- mcm4(cdc21-M68)-ts-dg::ura4+ ura4-D18 Our stock FY 6126 h+ cdc21-c106::kan ura4-D18 his 3D-1 leu1-32 ade6-M210 Our stock FY 6038 h- pcn1-K164R::ura4 cdc21-c106::kan ura4-D18 ade6-M210 Our stock 101 FY 6042 h+∆reb1::kanMX cdc21-c106::HphMx ura4-D18 leu1-32 ade6-M216 This work FY 6043 h- cdc21-c106::kan cyc17::ura4 his3 D-1 ade6- M216 ura4 cyc17=allelic to cig2 This work FY 6044 h+ cdc21-c106::kan cyc17::ura4 his3 D-1 ade6- M216 leui1-32 ura4 = allelic to cig2 This work FY 6052 h+ ∆rev1::ura4+ cdc21-c106::kan ura4-D18 his3-D1 ade6-M216/210?ura4-D18 This work FY 6053 h-∆rev1::ura4+ cdc21-c106::kan ura4-D18 his3? ade6-M?ura4-D18 This work FY 6054 h- eso1-eta ∆::kanMX6 cdc21-c106::HphMx ura4- D18 his3-D1 ade6-M210 This work FY 6055 h- eso1-eta ∆::kanMX6 cdc21-c106::HphMx ura4- D18 his3-D1 ade6-M210 Leu1-32 This work FY 6077 h+ ∆rad8::hphMX cdc21-c106::kan ura4-D18 his3-D1 ade6-M216 leu1-32 This work FY 6078 h- ∆brc1::ura4+ cdc21-c106::kan ura4-D18 ade6-M210 This work FY 6079 h-∆brc1::ura4+ cdc21-c106::kan ura4-D18 his 3-D1 ade6-M210 This work FY 6080 h+ ∆Ubc13::ura4+ cdc21-c106::kan his3D18 ura4-D18 ade6-m210 This work FY 6123 h- cdc21-c106::kan rev3::hphMX6 ura4-D18 his3-D1 ade6-M210 This work FY 6146 h+srs2::kan cdc21-c106::HphMx ade6-M210 leu1-32 ura4-D18 his3-D1 This work FY 6147 h-srs2::kan cdc21-c106::HphMx ade6-M210 ura4- D18 his3-D1 This work FY6238 h+ cdc21-c106::HphMx ura4-D18 his3-D1 leu1- This work 102 32 ade6-M210 FY 6248 h+∆ mms2::leu2 cdc21-c106::kan ura4-D18 leu1- 32 his4-239 ade6-M26 This work FY 6266 h-cdc21-c106::kan rad11-Cerulean::hphMX rad22-YFP-natMX ura4-D18 leu1-32 ade6-M210 This work FY 6281 h-cdc21-c106::kan chk1HA(ep) ade6-M216 ura4- D18 leu1-32 his 3D-1 This work FY 6308 h+ cdc21-c106::kan ∆cds1::ura4+ ura4-D18 leu1- 32 his3-D1 ade6-M210 This work FY 6309 h- cdc21-c106::kan ∆cds1::ura4+ ura4-D18 his3- D1 ade6-M210 This work FY6750 h- cdc21-c106::kan leu1-32::hENT1- leu1+(pJAH29) his7-366::hsv-tk-his7+(pJAH31) ura4-D18 ade6-M216 This work FY 6751 h+ cdc21-c106::kan mad2D::ura4+ ade6-M210 leu1-32 ura4-D18 This work FY 6777 h- cdc21-c106::HphMx ∆f1:kanMX6-Bioneer ura4-D18 ade6-M210 his3-D1 This work FY 6778 H+ cdc21-c106::HphMx ∆f1:kanMX6-Bioneer ura4-D18 ade6-M210 his3-D1 This work FY 6779 h- cdc21-c106::kan exo1::ura4 ura4-D18 ura4-D18 ade6-M210 This work FY 6780 h + cdc21-c106::kan exo1::ura4 ura4-D18 ura4-D18 ade6-M210 This work FY 6961 h-swi6::ura4+ cdc21-c106::Kan leu1-32 ura4- (DS/E or D18?) ade6-M210 *can1-1* This work FY 7045 h+cdc21-c106::kan fml1::natMX4 ura4-D18 his 3D-1 leu1-32 ade6-M210/216? This work FY 7047 cdc21-c106::kan fml1::natMX4 ura4-D18 his 3D- 1 leu1-32 ade6-M210/216 This work 103 FY 7048 h- chp1::kanMX6-Bioneer cdc21-c106::HphMx his3-D1 leu1-32 ura4-D18 ade6-M216/210? This work FY 7102 h90 cdc21-C106::Kan his3-D1 ura4-D18 leu1- 32 ade6-M216 This work FY 7165 h- ∆mus81::KanMX cdc21-c106::HphMx ura4- D18 his3-D1 ade6-M210 This work FY 7166 h+ ∆mus81::KanMX cdc21-c106::HphMx ura4- D18 his3-D1 ade6-M210 This work FY 7461 h- mcl1-11 cdc21-c106::kan ade6-704 ura4-294 leu1-32 his3D-1 This work FY 7462 h+mcl1-11 cdc21-c106::kan ade6-704 ura4-294 leu1-32 his3D-1 This work FY7611 h+ rhp51::ura4+ cdc21-c106::kan ade6-704/ade 6-M210 leu1-32 ura4-D18 This work FY 7802 h+ ∆chl1::kanMX6-Bioneer cdc21-c106::HphMx his3-D1 leu1-32 ura4-D18 ade6-M210 This work FY 7922 h+ arg3+::ccr1N-mCherry((D817 aa1-275)::his5+ cdc21-c106::kan rad11-Cerulean::hphMX rad22- YFP-natMX ura4-D18 his5D leu1-32 ade6- M210 This work FY 7923 h- ∆rif1::ura4+ ∆swi1::KanMX ura4-D18 leu1-32 ade6-M210 his3-D1 This work FY 7924 h+ ∆rif1::ura4+ ∆swi1::KanMX ura4-D18 leu1-32 ade6-M210 his3-D1 This work FY 7925 h- ∆rif1::ura4+ ∆swi3::KanMX ura4-D18 leu1-32 ade6-M210 his3-D1 This work FY 7926 h+ ∆rif1::ura4+ ∆swi3::KanMX ura4-D18 leu1-32 ade6-M210 his3-D1 This work FY 3664 h+ mcm4-"chaos" ura4-D18 leu1-32 ade6-M210 Our Stock FY 8000 h+/- hht1-mRFP:kanMX his7+::lacI-GFP lys1+::lacO cdc21-c106-HphMx leu1-32 ura4- This work 104 D18 FY 8015 h+ ∆ctf8::kanMX6-Bioneer cdc21-c106::HphMx his 3-D1 ura4-D18 Leu1-32 ade6-M210 This work FY 8016 h- ∆ctf8::kanMX6-Bioneer cdc21-c106::HphMx his 3-D1 ura4-D18 Leu1-32 ade6-M210 This work FY 8017 h+ ∆ctf18::kanMX6-Bioneer cdc21-c106::HphMx ura4-D18 Leu1-32 ade6-M210 This work FY 8018 h- ∆ctf18::kanMX6-Bioneer cdc21-c106::HphMx ura4-D18 Leu1-32 ade6-M210? This work FY 8107 h+/-? cdc20-M10 cdc21-c106::kan ura4-D18 ade6- M210 leu1-32 his3-D1 (polε) This work FY 7808 h+ rad21-K1::ura4+ cdc21-c106::kan ura4-D18 leu1-32 ade6-M216 his7-366/his 3D-1 This work FY 8108 h+/- ? ∆rad22::ura4+ cdc21c-106::kan ura4-D18 leu1-32 his3-D1 arg3-D4 This work FY 5014 h+ pcn1-K164R::ura4 ura4-D18 leu1-32 ade6- M210 Our stock FY 5270 h+ kpa1∆::bleMX6 his4-239 ade6-M26 Our stock FY 3124 h+ ∆rhp18::ura4+ ura4-D18 leu1-32 ade6-704 Our stock FY 4415 h+ ∆reb1::kanMX ade6-M216 ura4-D18 leu1-32 Our stock FY 277 h+ cyc17::ura4 ade6-M216 leu1 ura4 cyc17 is allelic to cig2 HIroto Okayama FY 5401 h+ ∆rev1::ura4+ ura4-D18 his4-239 ade6-M26 Our stock FY 4937 h+ eso1::kanMX6 ura4-D18 leu1-32 ade-M210 FY 5142 h+ ∆brc1::ura4+ ura4-D18 leu1-32 ade6- M210* Mathew O’Connell FY 4938 h+ rev3::hphMX6 ura4-D18 leu1-32 ade6- M210 Our stock FY 2050 h+ srs2::kan ade6-M210 leu1-32 ura4-D18 Our stock 105 FY 5260 h- ∆ mms2::leu2 leu1-32 his4-239 ade6-M26 Our stock FY 5625 h+ ∆rad8::hphMX leu1-32 ura4-D18 ade6- M216 his3-D1 Our stock/ derived from Bioneer FY 4742 h- rad11-Cerulean::hphMX rad22-YFP-natMX leu1-32 ade6-M210 ura4-D18 (rad11=ssb1) Our stock FY 4611 h- chk1HA(ep) ade6-M216 ura4-D18 leu1-32 Our stock FY 1163 h- rad12::ura4+ ade6-M210 leu1-32 ura4-D18 Our stock FY 3845 h-leu1-32::hENT1-leu1+(pJAH29) his7-366::hsv- tk-his7+(pJAH31) ura4-D18 ade6-M216 Our stock FY 1257 h+ mad2D::ura4+ ade6-M210 leu1-32 ura4-D18 Shelly Sazer FY 5583 h+ ∆rif1:kanMX6-Bioneer leu1-32 ura4-D18 ade6-M210 his3-D1 Our stock /derived from bioneer FY 3884 h- exo1::ura4 ura4-D18 Mathew O’Connell FY5555 h- ∆fml1::natMX4 ura4-D18 his3-D1 leu1-32 Our stock FY 4581 h- chp1::kanMX6-Bioneer leu1-32 ura4-D18 ade6-M216his3-D1 Our stock FY4159 h+ ∆mus81::KanMX Our stock FY1191 h- mcl1-11 ade6-704 ura4-294 leu1-32 (ts) Dwight Williams FY 1203 h+ rhp51::ura4+ ade6-704 leu1-32 ura4-D18 Greg Freyer FY 1318 h+ rec8::ura4+ ura4-D18 leu1-32 ade6-M210 Our Stock 106 FY 1159 h- rad21-K1::ura4+ ura4-D18 leu1-32 ade6-M216 his7-366 Our stock FY 3588 h- arg3+::ccr1N-mCherry((D817 aa1-275)::his5+ ura4-D18 his5D Zach Cande /XieTang FY 3227 h+ ∆swi1::KanMX ura4-D18 leu1-32 ade6-M210 his3-D1 Our stock FY 3228 h+ ∆swi3::KanMX ura4-D18 leu1-32 ade6-M210 his3-D1 Our Stock FY 7995 h- /+ arg3+::ccr1N-mCherry((D817 aa1- 275)::his5+ rad11-Cerulean::hphMX rad22-YFP-kanMX ura4-D18 his5D leu1-32 This work FY 5787 h+ hht1-mRFP:kanMX his7+::lacI-GFP lys1+::lacO leu1-32 ura4-D18 Our stock FY 7653 h+ ∆ctf8::kanMX6-Bioneer his 3-D1 ura4-D18 Leu1-32 ade6-M210? Our stock/ bioneer derived FY 8017 h+ ∆ctf18::kanMX6-Bioneer cdc21-c106::HphMx ura4-D18 Leu1-32 ade6-M210 Our stock/ bioneer derived FY 8110 h+/-?psf2-209 cdc21-c106::Kan ura4-D18leu1-32 ade6-M216 This work FY 8111 h+/-? rad35-271 cdc21-c106::Kan ura4-D18 leu1-32 ade6-M216 This work FY 2711 h+ psf2-209 ura4-D18 ade6-M216 leu1-32 Our stock FY 3999 h+ rad35-271 allelic to dfp1 Our stock FY 8197 h- pol1-1 cdc21-c106::Kan ura4-D18 leu1-32 This work 107 Figure Legends: Figure 3.1: Viability of mcm4 mutants at 36° and under MMS treatment (A)Temperature sensitivity evaluated by 1:5 serially diluted cultures plated on YES (rich media) and grown at the indicated temperatures. Wild type (FY 528), rad3∆ (FY1106), mcm4 chaos (FY 3664), mcm4-dg (FY 3395), mcm4-4SA (FY5251), mcm4 2-73∆ (FY 5688), mcm4-c84 (FY4310), mcm4-c106 (FY 4311), mcm4-M68 (FY784). (B) MMS sensitivity evaluated by 1:5 serially diluted cultures plated on YES (rich media) as control and 0.003% and 0.005% MMS at 25°. Figure 3.2: mcm4-c106 has a defect in replication at permissive temperature (A) Wild Type (FY 528), mcm4-M68 (FY784), mcm4-c106 (FY 4311) strains transformed with pUR19N plasmid plated on media lacking uracil. Colonies observed after seven days of growth at 25°. (B) Colonies isolated from transformation (A) streaked to single colonies on media lacking uracil and grown at 25° for 5 days. (C) Wild Type (FY 528), mcm4-M68 (FY784), mcm4-c106 (FY 6126) transformed with pDblet plasmid plated on media lacking leucine. (D) ade6-M210 his3-D1 FY 1110 h+ pol1-1 ura4-D18 leu1-32 ade6-M210 Our stock FY 6039 h+Kpa1 ∆::bleMX6 cdc21-c106::kan his4- 239/his3-D1? ade6-M26 ?(kpa1) Our stock FY 6040 h+∆rhp18::ura4 cdc21-c106::kan ura4-D18 leu1- 32 ade6-704 This work 108 Colonies isolated from transformation (C) streaked to single colonies on media lacking leucine, and grown at 25° for 5 days. Figure 3.3: Replication dynamics in mcm4-c106 at 36° and MMS (A) Relative viability of cultures during incubation at 36°. The indicated cultures were plated at 25° on YES plates; and viability was compared to the starting culture. (B) Bulk DNA content measured by flow cytometry of Sytox Green labeled cells. Cells were synchronized in G1 by nitrogen starvation and released to 25°, 36° and 0.01%MMS nitrogen-containing medium. Wild Type (FY261), mcm4-c106 (FY 4311). (C) Pulsed field gel electrophoresis (PFGE) analysis in each genotype in untreated asynchronous (AS) cells, after 4h 36° (4h) and after 2h release to 25° (R).Wild type (FY 528), mcm4-M68 (FY784) mcm4-c106 (FY 4311). S. pombe chromosomes are indicated on the left (D) Relative viability of cultures during incubation with 0.01% MMS. The indicated cultures were plated at 25° on YES plates; and the viability was compared to the starting culture Figure 3.4: mcm4 protein levels and Chk1 phosphorylation in response to MMS and 36°. (A) Evidence for Chk1 activation following 0.01% MMS for 4hr. Chk1 mobility in SDS-PAGE was used as a proxy for phosphorylation. Lane 1,2 – mcm4-c106 (FY 4311; no Chk1 tag) lane 3,4 – chk1-HA (FY 4611), lane 5,6 mcm4-c106 chk1-HA (FY6281). Arrow indicates phosphoshift. (B) Activation of Chk1 following 4hr incubation at 36°. Lane 1,2 - mcm4-c106 (FY 4311), Lane 3,4 chk1-HA (FY 4611), Lane 5,6 mcm4-c106 chk1HA (FY6281). (C) Mcm4 protein levels after MMS treatment. (D) Mcm4 protein levels after incubation at 36°. Figure 3.5: Accumulation of repair foci in mcm4-c106 in response to MMS and temperature 109 (A)RPA and Rad52 focus patterns during treatment at restrictive temperature (36°) and release. Multiple small foci were observed with the mcm4-c106 which remained after release. RPA and Rad52 focus patterns during treatment with 0.01% MMS for 4hrs and release for 2hrs. Multiple small foci were observed during treatment and release in the mcm4-c106 compared to the wild type (B) Quantification of Rad11 foci of Wild Type and mcm4c106 during 36° treatment and release (C) Quantification of Rad52 foci of Wild Type and mcm4c106 during 36° treatment and release .(D) Quantification of Rad11 foci of Wild Type and mcm4c106 during 4hr MMS treatment and release (E) Quantification of Rad11 foci of Wild Type and mcm4c106 during 4hr MMS treatment and release Figure 3.6: rif1∆ rescues the mcm4-c106 MMS phenotype: (A)Serial dilutions of rif1∆ mcm4-c106 on YES at 25°, 32°, 36° (B) Serial dilutions of rif1∆ mcm4-c106 (C) swi1∆ mcm4-c106 and swi3∆ mcm4-c106 on YES at the indicated MMS concentrations grown at 25° Figure 3.7: mcm4-c106 interactions with alternative replication factor C (RFC): (A) mcm4-c106 combined with RFC Ctf18 ∆ and RFC Ctf8 ∆. Representative response to temperature was assayed by serial dilutions. Strains were grown overnight at 25° and 1:5 serially diluted and plated on YES (rich media) as the control and 32° and 36° to observe the temperature effect. (B) Representative response to MMS assessed by serial diluted samples plated on the indicated concentrations of MMS (C) chl1∆ mcm4-c106 (D) cohesion subunit rad21-K1 mcm4-c106 effects on MMS 123 Supplemental Figure legends: Figure S1: Viability of mcm4 mutants treated with HU and CPT HU and CPT sensitivity evaluated by 1:5 serially diluted cultures plated on YES (rich media) and grown at 25°. Wild type (FY 528), rad3∆ (FY1106), mcm4-M68 (FY784) , mcm4-dg (FY 3395), mcm4-c106 (FY 4311), mcm4-c84 (FY4310) ,mcm4-4SA (FY5251), mcm4 2-73∆ (FY 5688) Figure S2: mcm4c-106 chromosome segregation during mitosis at 36° using LacI LacO system Wild Type (FY5787) and mcm4-c106 (FY 8000) were asynchronously grown at 25° over night to early log phase (~0.3-0.4) and shifted to 36° for 4hrs and chromosome segregation was observed in real time. Figure S3: Effects of Cig2∆ on mcm4-c106 MM S sensitivity MMS sensitivity was evaluated using a Cig2∆ mcm4-c106 double mutant plated on indicated concentrations of MMS Figure S4: mcm4-c106 interactions with genes involved in the error free and error prone repair pathways (A) MMS sensitivity of double mutants generated with genes involved in the error free pathway plated on the indicated concentrations of MMS and grown at 25°. (B) Serial dilutions of mcm4- c106 combined with genes involved with the error prone pathway plated on indicated MMS concentrations grown at 25° Figure S5: mcm4-c106 interactions with genes involved in recombination and other pathways Indicated strains were serially diluted and plated on indicated concentrations of MMS to evaluate the sensitivity 124 Chapter 4 Mini-chromosome maintenance complexes form a filament to remodel DNA structure and topology The work in this chapter was originally published as: Slaymaker IM, Fu Y, Toso DB, Ranatunga N, Brewster A, Forsburg SL, Zhou ZH, Chen XS. (2013) Mini-chromosome maintenance complexes form a filament to remodel DNA structure and topology. Nucleic Acids Res. 2013 Mar 1;41(5):3446-56. For the publication, I contributed towards invivo work that was done by performing yeast genetics assays using S.pombe to test the biological relevance of the α5 subdomain interactions. I used site directed mutagenesis to create the relevant point and truncation mutations, and cloned them to different vector systems to performed complementation assays. The results are indicated in Supplementary figure 8 and Table 2. Mini-chromosome maintenance complexes form a filament to remodel DNA structure and topology Ian M. Slaymaker 1 , Yang Fu 1 , Daniel B. Toso 2,3,4 , Nimna Ranatunga 1 , Aaron Brewster 1,5 , Susan L. Forsburg 1 , Z. Hong Zhou 2,3,4 and Xiaojiang S. Chen 1, * 1 Molecular and Computational Biology, University of Southern California, Los Angeles, CA 90089, 2 Department of Microbiology, Immunology, and Molecular Genetics, 3 Biomedical Engineering Interdepartmental Program, 4 California NanoSystems Institute, University of California, Los Angeles, CA 90095 and 5 Biological Sciences, 731 Stanley Hall, University of California, Berkeley, CA 94720-3220, USA Received August 28, 2012; Accepted December 21, 2012 ABSTRACT Deregulation of mini-chromosome maintenance (MCM) proteins is associated with genomic in- stability and cancer. MCM complexes are recruited to replication origins for genome duplication. Paradoxically, MCM proteins are in excess than the numberoforiginsandareassociatedwithchromatin regions away from the origins during G1 and S phases. Here, we report an unusually wide left-handed filament structure for an archaeal MCM, as determined by X-ray and electron micros- copy. The crystal structure reveals that an a-helix bundle formed between two neighboring subunits plays a critical role in filament formation. The filament has a remarkably strong electro-positive surface spiraling along the inner filament channel for DNA binding. We show that this MCM filament binding to DNA causes dramatic DNA topology change. This newly identified function of MCM to change DNA topology may imply a wider functional role for MCM in DNA metabolisms beyond helicase function.Finally,usingyeastgenetics,weshowthat the inter-subunit interactions, important for MCM filament formation, play a role for cell growth and survival. INTRODUCTION All organisms must duplicate their genome to provide each new cell with a full complement of genetic informa- tion prior to mitotic division. When a cell enters S-phase, double-stranded genomic DNA is separated into single strands to be copied into sister chromatids. This process is tightly regulated and highly coordinated to ensure the high-fidelity replication of the genome. EukaryoticandarchaealchromosomalDNAreplication are initiated by the stepwise assembly of pre-replicative complexes (pre-RCs), composed of the origin recognition complex(ORC),Cdc6andmini-chromosomemaintenance (MCM) complexes (1,2). These components cooperatively catalyze the initiation of replication at the origin. Upon origin firing, the MCM ring complex acts as an ATP- dependent DNA helicase to unwind the genome, opening double-stranded DNA (dsDNA) into single-stranded DNA (ssDNA) templates (3). Eukaryotes express six essential homologous MCM proteins (MCM2-7) that form hexamers and double hexamers in vitro (4–6). Archaeal genomes also encode MCM genes with sequence and structural homology to eukaryotic MCM2-7 (7). However, many archaea express only a single MCM subunit, which forms homo-oligomeric complexes with the same function as the eukaryotic MCM complexes (8–10). In G1 and leading up to S-phase of the eukaryotic cell cycle, multiple MCM2-7 hetero-hexamers are recruited to eachpre-RCattheoriginandspreadtonearbychromatin (11–13). Mutations that limit pre-RC to only a single iteration of MCM recruitment are not viable, which suggests that the recruitment of many MCM proteins is arequirementforproperpre-RCfunction(11,14–17).This is puzzling as only one or two MCM hexameric rings are sufficient to unwind DNA (8,18,19). How MCM functions when bound to chromatin prior to and during initiation has been the subject of much interest, though results often raise more questions than answers. The chromatin-bound MCM complexes are categorized into two biochemically distinguishable sub- groups. One is the salt-stable ‘loaded’ complexes that are bound tightly to the origin, likely locked onto DNA as hexamer or a double hexamer as the active helicase form in vivo (4,6). The other is the salt-sensitive ‘associated’ complexes and not specifically located at origins, which *To whom correspondence should be addressed. Tel:+1 213 821 1255; Fax:+1 213 740 4340; Email: Xiaojiang.chen@usc.edu 3446–3456 Nucleic Acids Research, 2013, Vol. 41, No. 5 Published online 29 January 2013 doi:10.1093/nar/gkt022 Published by Oxford University Press 2013. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/ by-nc/3.0/), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. accounts for the majority of the MCM proteins. These MCM proteins distal from the origin may have different biological function(s) (12,20–22). MCM proteins have been detected associating with large regions of unreplicated chromatin during G1 and early S-phase (13). Despite the large number of MCM proteins in the nucleus, reduction in MCM gene dosage causes genome instability, demonstrating the importance of maintaining MCM protein level for cell survival (23–26). These peculiarities are part of what has been termed the ‘MCM paradox’ [reviewed in (27) and refer- ences therein]. In this study, we report a crystal structure of the near full-length MCM from the archaeon Sulfolobus solfataricus (sso) assembled as a wide helical filament with a large channel along the filament axis. We also have verified the formation of the same filament structure in solution using electron microscopy (EM). The crystal structurerevealedastructural elementcritical forfilament formation, which has been confirmed by structure-guided mutagenesis and EM studies. Furthermore, we used yeastgeneticstoshowthatthisstructuralelement,import- ant for filament formation, is critical for cell growth and survival. METHODS ssoMCM cloning and MCM purification Wild-type (WT) full-length ssoMCM containing residues 1–686 are cloned as His-tagged fusion proteins in vectors pGEX-6P-1. Mutants of MCM were made using either Quikchange or PIPE (28) and expressed as previously described (9). Full-length WT and mutant MCM proteins were purified from Escherichia coli grown at 25 C for 18h. Cells were lysed by French-press and centrifuged at 10000rpm for 1h. Supernatant was passed over nickel resin and washed with 10 column volumesofhigh-saltBufferA(1MNaCl,50mMHEPES, pH 7.5). MCM was eluted with five column volumes of Buffer A supplemented with 250mM imidazole. Eluted MCM fractions were diluted or dialyzed to 100mM NaCl and passed over a Resource Q column. MCM was eluted from the Resource Q by a 10-column volume salt gradient from 50 to 500mM NaCl. Resource Q fractions were diluted to 100mM NaCl and passed over a heparin column. MCM was eluted from the column with a 50–1000mM NaCl linear gradient. Heparin fractions were collected, concentrated to 1 ml in 250mM NaCl, 20mM HEPES, pH 7.5, 2mM DTT and then further purified by Superdex 200 column chromatography. Superdex 200 fractions were concentrated to 30–50mg/ ml and flash-frozen in liquid nitrogen. Crystallization and structure determination To assemble a homogeneous complex, MCM (50mg/ml) was initially incubated with a 61-bp dsDNA with a 3 0 -T overhangs on each strand and dialyzed from 2M NaCl to 50mM overnight at 4 C for crystallization trays. DNA strands for annealing into the 61-bp dsDNA (strand1:tagctattagagcttggtttaattatacaaactcaatatttttcttttttc cttcctttat, strand2:tatcgataatctcgaaccaaattaatatgtttgagtta- taaaaagaaaaaagaaggaaat) were purified using a MonoQ column (GE), annealed overnight and further purified on a Superdex 200 gel filtration column (GE). Hanging- drop crystal trays were set up at 4 C. Small but long needle crystals grew to dimensions of 3040200mm at 4 Cin2ml drops with ratios of 1–1.5ml MCM to 1–0.5ml of crystallization buffer (7.5% isopropanol, 420mM NaSO 4 and 20mM HEPES, pH 6.75). Crystals were harvested, cryoprotected in 420mM LiSO 4 , 25% PEG 400, 25mM HEPES, pH 6.75 and flash-frozen in liquid nitrogen. Data were collected at APS beamline GMCA/CAT 23-ID (and 19-ID), using the 5-mm microbeamvectoredoverthelengthofthecrystalneedles. Diffraction spots were detected up to 3.8A ˚ with 30-s exposure at 23-ID. However, a 30-s exposure time essentially kills the crystal diffraction. As a result, we used a combination of 5-s exposure time, microbeam, translations every three to five exposures along the needle crystals and multiple crystals to collect data suffi- cient for obtaining the highest resolution data set to 4.29A ˚ . Later on, a different crystallization condition (0.1M HEPES, pH7.0, 18% MPD) yielded a crystal form that has the same space group and same filament structure, but are more resistant to radiation damage up to 120-s exposure time in 23-ID-D and we are able to extend the resolution of the structure to 4.1A ˚ (Supplementary Table S1). The crystal is in space group P21, with five subunits per asymmetric unit. Thestructurewasdeterminedbymolecularreplacement using3F9Vwithloopstrimmedoffasasearchmodelusing the Phaser program in the Phenix suite (29). R-free flags (5%) were set at this point and communicated between Phenix and CNS as needed. The initial model was rebuilt asapolyalaninestructureinCootwithreiterativeroundsof solvent flipping and solvent flattening using CNS, density modification and 5-fold non-crystallographic symmetry (NCS) averaging from the CCP4 suite or CNS. Once the main chain was properly placed, side chains were added and refined using CNS 1.3 deformable elastic network (DEN) low-resolution refinement strategies (30). The ref- erence model for DEN restraints was a hybrid of the N-terminal ssoMCM and homology model of mkaMCM threaded through the structure model from 3F9V. The DEN refinement improved the phases at this resolution, as evident by the improved density and correct side-chain positioning,whichisthecasefortherefinementofanother large complex structure with 5.0A ˚ data (31). After DEN refinement, the model was further rebuilt by reiterative rounds of density modification in CNS and model building in Coot using B-factor sharpening and Density Modification in CNS (32). Further model improvements were made using phenix.refine in Phenix version 1.7.2-863 (29).The5-foldNCSrestraintswereimposedatallstages. Finalrefinementswithgeometryrestraintsweredoneusing Refmac and phenix.refine imposing secondary structure restraints, TLS restraints, Ramachandaran restraints and 5-foldNCStoafinalmodelR-work/R-free34.38/35.25and Ramachandaran statistics 85.3% in the most favored and 0.8% outliers. Identical set of reflections were used for R- free at all stages ofrefinement (Supplementary Table S1). NucleicAcidsResearch,2013,Vol.41,No.5 3447 Once the model was refined satisfactorily, validation and final statistics were acquired using Molprobity server (http://molprobity.biochem.duke.edu/) and phenix.validate (29) (Supplementary Figure S1d). Our structure fell into the 83rd percentile among structures from 3.25 to 4.36A ˚ resolution with 0 bad bonds, 0 bad angles and excellent statistics among structures of similar resolution.N-terminalresidues1–6arenotincludedinthe structure as no density was seen. Density for the flexible C-terminal 88 residue wing-helix domain of ssoMCM was also not visible and thus the final model contains residues 7–598 for each of the five subunits in the asymmetric unit or half of one helical turn. Although broken density was observed within the central channel, we were unable to build a model for dsDNA into the filament. Transmission electron microscopy and electron tomography Negatively stained samples were prepared by placing a small drop (4ml) of sample solution onto a glow- discharged carbon-coated copper grid. For transmission electron microscopy (TEM) and electron tomography (ET), 200-mesh and 100/400 slotted grids were used, re- spectively. After a period of 1 min at room temperature, the sample was blotted and a drop of 2.5% uranyl acetate solution was immediately placed on the grid. After staining for 1 min the drop was blotted off, the grid was washed four times with the same stain solution and then allowed to air dry. ThestainedsampleswerevisualizedwithanFEITecnai F20transmissionelectronmicroscopewithanaccelerating voltage of 200kV. The samples were imaged at 50000–100000 with an underfocus value of 3mm. Tomography tilt series were taken using the FEI Batch Tomography software with a tilt range from 70 to +70 . The tilt series were recorded on a 16-megapixel TVIPS CCD camera. Alignment of the tilt series was performed using the ‘etomo’ tomography processing software from the Imod package (33). The steps included removing X-ray hot spots, rough alignment by cross-correlation and fine alignment by patch tracking. The aligned tilt series were thenusedtomakethree-dimensional(3D)reconstructions using GPU-based SIRT (simultaneous iterative reconstruction technique) reconstruction implemented in ‘Inspect3D’ (FEI). Slices from the 3D tomography maps were displayed using the ‘slicer’ tool within the ‘3dmod’ program of the Imod package. Amira (Visage Imaging GmbH, http://www.amira.com/) was used to segment and to create volume renderings of the 3D density maps of the filaments. Consurf, sequence alignments and electrostatics analysis Conserved region alignments and coloring were done through the consurf server (http://consurftest.tau.ac.il/) (34,35). Multiple protein alignments were done with the ClustalW server (http://www.ebi.ac.uk/Tools/msa/ clustalw2/) (36,37). Electrostatics of MCM filament struc- ture were calculated using the APBS plug-in as part of Pymol 1.4 (38). Assays for DNA binding, helicase activity and oligomerization A range of concentrations of purified MCM protein was incubated with 0.2mM 61-bp dsDNA in binding buffer (10mM Tris pH 8.0, 50mM NaCl) at room tem- perature for 30min. 10ml reactions with 5% glycerol were electrophoresed (in 0.5% agarose, 0.5 TBE) at 90V for 40min. After electrophoresis, gels were stained in ethidium bromide and visualized under UV light. The DNA binding was quantified using Quantity One software. Helicase assays were performed as previously described (39). For oligomerization assay, purified MCM and MCM mutants were dialyzed in a buffer containing 10mM HEPES, pH 7.5, 50mM NaCl and 2mM DTT. 500mg proteinin100mlwasanalyzedbygelfiltrationchromatog- raphy on an analytical Superose 6 column at 4 Cina buffer containing 10mM HEPES, pH 7.5, 250mM and 2mM DTT. DNA topology footprints MCM was dialyzed into a buffer containing 50mM NaCl and10mMTris8anddilutedto6mg/ml.A15-mlreaction solution containing 500ng of plasmid DNA (pBR233, New England Biolabs) and MCM was incubated at room temperature for 30min. About 5U of E. coli TopoisomeraseIwereaddedtothereactionandincubated for3hat37 C.Aquantityof25mMEDTAand5%SDS were added to stop the reaction which was then deproteinated by addition of proteinase K. Samples were run on a 1% agarose gel either with or without 1.4mg/ml of the intercalator chloroquine added. Yeast plasmid and mutation construction for yeast genetics Nucleotide changes to introduce the point mutation and the internal deletion were created using the Phusion site-directed mutagenesis kit (New England Biolabs) following the manufacturer’s instructions. The constructs were sequenced (Laragen and Genewiz) to confirm the presence of the mutation and to confirm that PCR mutagenesis did not introduce additional mutations. Yeast strains and manipulations Fission yeast strains used for the study were grown in yeast extract plus supplements or in Edinburgh minimal medium (EMM) with appropriate supplements. In this work, WT strain refers to FY 261 (h+ can1-1 leu1-32 ade6-M216 ura4-D18) and mcm4 ts refers to FY 784 (h+ cdc21-M68 ura4-D18 leu1-32 ade6-M210 can1-1). Yeast plasmids used for this work are derived from REP82X and contain a ura4+marker, the weakest nmt promoter and a HA tag at the C-terminus. pNR29 served as the WT (positive control), whereas pSLF372 served as the vector only (negative control). Transformations were carried out by electroporation and candidates were selected on EMM media lacking uracil, which also contained 15mm thiamine for full repression of the 3448 NucleicAcidsResearch,2013,Vol.41,No.5 nmt promoter. Plates were allowed to grow at 25 C for 7 days following the transformation or until colonies were visible. Once colonies were present, complementation analyses of WT and mcm4 temperature-sensitive strains were carried out by streaking six independent colonies from each transformation on EMM media lacking uracil, supplemented with 15mM thiamine and incubated at 25, 32 and 36 C for 7 days. Plates were scanned on Days 3, 5 and 7. The represented figures are following 5 days of incubation at the designated temperatures. RESULTS MCM filament crystal structure Using a full-length MCM protein from the archeaon ssoMCM, crystals were obtained in the presence of 61- bp dsDNA, but not in the absence of dsDNA, suggesting dsDNA is an integral part of the crystal, which is also confirmed from agarose gel analysis of the solubilized crystals. The crystal structure is an unusually wide left-handed filament (Figure 1a), with 10 subunits per helical turn. Each asymmetric unit (asu) contains five MCM subunits. Despite the presence of DNA in the crystals, we can only detect non-featured extra density along the filament channel that could be accounted by the bound DNA with some freedom of rotation/sliding in each asu. Although full-length MCM protein was used in crystallization, density of a small wing helix-like domain at the C-terminus was not visible, and thus the final model contains MCM protein residues 7–598 and is missing residues 599–686. Even though the resolution of the diffraction data goes to 4.1A ˚ , the recently developed refinement methods [DEN refinement (30), also see ‘Methods’ section] worked well to enable the confident placement of side chains in the electron density map (Supplementary Figure S1a, b and c), with excellent geometry and statistics (Table 1). The filament structure reveals an outer diameter of 175A ˚ and a large inner channel opening of 90A ˚ (Figure 1a and b). The structure shows a narrow filament groove. Parallel to the narrow groove is a furrow on the filament outer surface (Figure 1a) with side channels formed between neighboring subunits which connect to the interior of the filament central channel (Figure 1a). To exclude the possibility that the filaments are a crys- tallographic artifact, we used ET to detect the oligomeric state of MCM bound to dsDNA in solution. The same (a)(b) (c) (d) Figure 1. Overall features of the MCM filament structure. (a) Surface representation of the crystal structure of a filament of full-length ssoMCM. The left-handed filament contains 10 subunits per turn. (b) Top–down view through the filament central channel, with dimensions indicated. (c) View of a MCM monomer (ribbon in green) in the filament. Regions playing important roles in making contacts in the filament are labeled. (d) Monomer structure with a neighboring subunit in the filament shown in gray. The division of the N-domain and C-domain and the subdomains (A, B, C, N–C linkers, etc.) are indicated. Notable structural features are labeled: Nt-hp; PS1 (pre-sensor 1), EXT (external hairpin), PH3-lp (pre-helix 3 loop), P-hel (P-loop helix), P-lp (P-loop), H2I (helix-2 insert), ACL (allosteric communication loop). The ATP binding pocket is marked with a red asterisk. Table 1. X-ray refinement statistics Resolution (A ˚ ) 4.1 R work /R free 33.14/34.13 Number of atoms 23590 Protein 23585 Ligand/ion 5 Water B-factors Protein 197.65 Ligand/ion Water RMSD Bond lengths (A ˚ ) 0.008 Bond angles ( ) 0.768 NucleicAcidsResearch,2013,Vol.41,No.5 3449 type of filaments were observed when MCM protein was pre-incubated with naked dsDNA in solution but not when incubated with ssDNA or in the absence of DNA (Figure 2a). The 3D ET reconstructions of filaments revealed that the filament groove and pitch, dimensions and handedness, matches those of the crystal structure well, as shown in Supplementary Movie S1. A prior EM study reported a very thin right-handed helical filament of a different archaeal MCM (mtMCM) (10). We were unable to detect any thin right-handed filament forms by EM using either mtMCM or ssoMCM, in varying buffer conditions, with or without DNA or added nucleotides (40). However, when we were revising this article, a left-handed lock washer structure (with filament arrangement) for MCM2-7 was published (41), providing an example for a left-handed structure for an initiator protein, such as MCM. The structure of the MCM monomer in the filament is shown in Figure 1d, which consists of two separable domains, an N-terminal domain (containing A, B and C subdomains) and the C-terminal domain [containing AAA+, a–helix 5 (a5) and a-subdomains], joined by a long linker (N–C linker). Despite a similar overall core fold to the previously published monomeric structure of ssoMCM [3F9V (9)], the structure in the context of the filament has some obvious conformational differences, as reflectedbyanRMSDof2.5A ˚ 2 forthesuperimpositionof the two structures. Compared with the previous structure, the C-terminal domain is rotated 17 about the N–C linker and swung away from the side facing the central channel. Within the C-terminal domain, an a5 helix (Figure 3a) rotates dramatically (90 rotation) to take a different position and orientation, which has significant consequences in filament assembly. At the N-terminus, the zinc-bearing B subdomain also has some positional shifts, revealing a structural flexibility about the b-sheet bridging the B- and C-subdomains (8,9). Within the N-terminal half, another noteworthy difference from the previous ssoMCM structure is that the long N-terminal hairpin (Nt-hp) from the filament has a large shift to point in a different direction (Figure 4a), which alters the surface charge features dramatically, generating a spiraling charged surface differing from that of a hexamer with a horizontal changed surface within a ring (Figure 4b). The new conformations of these structural (a)(b) (c)(d) Figure 2. ET and TEM imaging of MCM–dsDNA filament. (a) Side-by-side comparison of the MCM filament crystal structure and ET reconstruction. The crystal structure is filtered to 8A ˚ (left) and a central slice from the ET reconstruction is shown to the right. Note that the shadow in the EM image makes it to appear wider. The superimposition of the crystal structure over the ET reconstruction image shows a well-matched groove dimension and handedness (Supplementary Movie S1). (b) An ET reconstruction of MCM filament showing DNA protruding from the ends (also Supplementary Movie S2 for views of different sections). (c) Electron micrograph of WT MCM filament on 1000-bp linear dsDNA, forming a filament with 175A ˚ thickness, but a small fraction of it (bracket) showing filament with similar thickness obtained from a mutant shown in panel d. Approximately 1/100 of the wide filaments from WT MCM contains such a small portion of thin filament. (d) Electron micrograph of a5-linker mutant on 1000-bp linear dsDNA, exclusively forming a filament with 125A ˚ thickness. Unlike the WT MCM, this mutant cannot induce supercoiling of plasmid DNA (also compare Supplementary Figure 2a with c and Supplementary Figure 4a with b). Black scale bars are 500A ˚ . Figure 3. The helix a5 rotation and its interaction with the a-subdomain of a neighboring subunit in the filament structure. (a) C-domain structural alignment between the filament subunit (fila-ssoMCM, green) and the previously determined monomer struc- ture (3F9V, mon-ssoMCM, yellow). Fila-MCM helix-5 (a5) is rotated 90 (red) relative to the mon-ssoMCM a5. (b) a5 (red) of one subunit (mon2) docks on the a-subdomain (blue) of a neighboring subunit (mon1), forming a four-helix bundle between mon1 and mon2 in the filament structure. The EXT hairpin (cyan) is just above the 4-helix bundle. (c) The side chains (in stick model) of some of the critical residues at the interface between a5 and a-subdomain as well as on the a5-linker for regulating filament formation (Figure 2d and text). 3450 NucleicAcidsResearch,2013,Vol.41,No.5 elements appear to be important for forming contacts in filament formation, as discussed below. dsDNA binds within the MCM filament channel As only broken and not featured extra density was seen within the filament channel, which probably is due to the lack of fixed positioning of DNA, it was not possible to build the model for the 61-bp dsDNA in the co-crystal. Using ET to examine the MCM protein incubated with 1000-bp dsDNA, however, the dsDNA was detected with the same form of MCM filament, with dsDNA clearly visible protruding from both ends of the filament channel in EM tomographs (Figure 2b). The different Z-sections of DNA-bound filament are revealed in Supplementary Movie S2, which shows that the filament features match those of the crystal structure. Electrostatic analysis of the crystal structure revealed a remarkably charged inner surface that is highly electro-positive and forms a long, continuous ‘blue’ (electro-positive) strip along the filament inner channel surface (Figure 5a–d), immediately suggesting a role in DNA binding. Three pairs of residues, K246/R247 of one subunit, and R379/K381 and K408/R410 of an adjacent subunit, cluster on the filament channel interface toformthiselectro-positivestripinthefilament(Figure5c and d). To confirm that this electro-positive surface of the filament binds DNA, we mutated each residue pair to Ala and assayed their DNA-binding activity. As predicted, K408A/R410A mutant completely abolished dsDNA binding (Figure 5e). R379A/K381A and K246A/R247A mutants individually caused slight decrease in dsDNA affinity, and when combined dsDNA binding was com- pletely abolished (Figure 5e). These results confirm the DNA-binding role for this spiral electro-positive strip on the filament channel surface. Examination of these mutants by EM revealed no filament formation, further illustrating the need for binding DNA for the filament formation. MCM filament induces negative DNA supercoils Knowing that the left-handed MCM filament can bind lineardsDNAinpackedcrystalandinsolution,wesubse- quently used EM to examine MCM binding to closed circular plasmid dsDNA. The result revealed that the plasmid DNA coated by WT MCM filaments became heavilysupercoiled(SupplementaryFigureS2a),indicating MCMcaninducetopologicalchangestoDNA. To further confirm that MCM generates and stabilizes negative supercoils, we used a topology footprinting assay to observe changes in DNA topology as described previously(42,43).Inthisassay,we addedTopoisomerase I to plasmid DNA bound by various amount of MCM to nick the circular DNA backbone and relieve topo- logical stress of supercoils introduced by MCM binding, allowing DNA to relax into the lowest energy topoisomer that was then stabilized by ligation. Proteins were then degraded by proteinase K to isolate the stabilized circular DNA with the altered linking number. The results demonstrated that MCM generated the negatively supercoiled topoisomers of plasmid DNA in a dose- dependent manner (Figure 4c). To confirm that the observed DNA topology change induced by MCM is indeed negatively supercoiled, the same footprinting reactions were performed, but the reac- tions were analyzed on agarose gels containing chloro- quine, as chloroquine can bind dsDNA and change the relative mobility of topoisomers depending on the negative or positive supercoiling, allowing a differenti- ation between negative and positive supercoiling (43). The result showed that presence of chloroquine on the agarose gel shifted the MCM-induced supercoiled DNA toward the sample wells relative to the chloroquine-free gels (compare Supplementary Figure 4b and c), confirm- ing that changes in DNA topology induced by MCM in the assay were due to a negative change in linking number (Lk). A closer examination of the filament crystal structure revealed that the previously mentioned positively charged residue pairs (such as K246/R247) critical for DNA binding are spaced periodically 26A ˚ apart along the left-handed electro-positive strip (Figure 5f), which is similar to the groove periodicity of A-form DNA (24.6A ˚ / turn,150A ˚ inlengthfora61-bpDNA),butverydifferent from that of B-form DNA (34.5A ˚ /turn,210A ˚ in length for a 61-bp DNA). Although we cannot confirm that A-form DNA is indeed present due to a lack of defined DNAelectrondensityinthecrystalstructure,weusedmo- lecular modeling to position the 61-bp DNA on the Figure 4. The N-terminal domain structural alignment and the DNA topology change induced by MCM. (a) Alignment of MCM N-terminal structures, showing the conformational change for the Nt-hp in the filament structure (green), with the Nt-hp pointing to a different direc- tion to make contact with a neighboring subunit. Monomeric mon-ssoMCM in yellow (3F9V), N-ssoMCM in orange (2VL6), N-mtMCM in purple (1LTL). (b) Electrostatic patterning of ssoMCM filament (five subunits) compared with electrostatics of a hexamers of the N-terminal ssoMCM. The Nt-hp conformation in the filament structure reconstitutes an electro-positive surface (blue surface) to follow the spiral path in the filament. The orange objects represent the DNA with the expected DNA binding orientation on the filament or the hexamer. (c) DNA topology footprint with MCM, showing more negative supercoiling of DNA induced by increasing amount of MCM. Lane 1: negatively supercoiled pBR233 plasmid; lane 2: relaxed (nicked by topoisomerase) plasmid DNA and lanes 3–9: relaxed plasmid DNA incubated with increasing concentrations (1–10mM) of MCM. OC, open circle; () SC, negative supercoiling; Lk, linker number. NucleicAcidsResearch,2013,Vol.41,No.5 3451 electro-positivestripofthefilament,whichwouldrequirea transitionfromB-formtoA-formtofitboththeperiodicity of the 26A ˚ and the 153A ˚ unit length per asu (Figure 5f and g). The transition from B-form DNA to A-form requires untwisting (or net negative twist) of the right- handed duplex DNA, loosening the double helix. Such a local loosening of the right-handed double-helix intro- duced through binding to the left-handed MCM filament will generate supercoiling of a circular plasmid DNA in order to compensate for the local untwisting of the duplex, thus providing a structural basis for the observed supercoiling of DNA induced by MCM. MCM helix a5 regulates oligomerization Spatial alignment of the AAA+subdomains with the pre- viouslysolved ssoMCM monomer structure (9)revealeda 90 rotation of a5 (Figure 3a). This rotation brings a5 close to the three helices of the a-subdomain of a neigh- boring subunit, allowing a5 to form an inter-subunit four helix bundle with the a-subdomain in the filament struc- ture (Figure 3b and c). Linkers flanking a5(a5-linkers) contain Gly and Pro, which can provide the flexibility as wellasrigidityofthelinkerstoenablea5tohavethelarge rotations necessary for interacting with the a-subdomain of another subunit in the filament form. Additionally, Figure 5. The strong electro-positive ‘strip’ along the helical filament inner surface for DNA binding. (a) Spiral positively charged electrostatic pattern (the blue strip) along the inner wall of the helical MCM filament. (b) End view of the filament looking down the filament central axis, showing the spiral electrostatic pattern (the blue strip) down the central channel. (c) A half turn of the filament (five subunits in one asymmetric unit or asu), showing the electrostatic potential surface at 1 (red mesh) and 1 (blue surface) Kt/e. (d) Arrangement of the six positively charge residues (or three pairs) on the electro-positive strip. (e) Mutational effects of residues on the positively charged strip for dsDNA binding. (f) Top–down view along the filament channel, showing the periodicity (26A ˚ ) of positively charged residues of each subunit and the length (153A ˚ ) of the electro-positive surface along the filament in one asu (five subunits), which match the groove periodicity (24.6A ˚ ) and the length (150A ˚ ) of a 61-bp DNA in A-form, as shown in panel g. (g) The 61-bp A-form dsDNA modeled on to the electro-positive strip over a path of five subunits of the filament, showing charged protein surface and matching periodicity of groove spacing of the A-form DNA. ND, none detected. 3452 NucleicAcidsResearch,2013,Vol.41,No.5 some residues at the binding interface between a5 and a-subdomain helices are highly conserved among archaeal and eukaryotic MCMs (Supplementary Figure S5). Thus, we predicted that not only the Gly/Pro residues of the a5-linkers but also the conserved residues (such as F540, Supplementary Figure S7a)at theinterface between a5 and a-subdomain are important for proper inter-subunit interactions for filament formation. To test this, we created mutants of the conserved residues on the interface between a5 and a-subdomain (F540A mutant) and of the Gly and Pro residues on the a5-linker (G485P/G501P mutant). By gel filtration chromatog- raphy, the G485P/G501P and F540A mutant proteins eluted as smaller oligomers (with a molecular weight similar to a trimer) than the WT oligomeric form (a hexamer form) in 250mM NaCl buffer (Supplementary Figure S6a), clearly indicating compromised subunitasso- ciation, even though not a complete disruption. Wealsoexaminedwhetherthesemutationswouldaffect filament formation. By EM examination, we found that F540A mutant could not form any filaments, but formed only closed or open ring circular structures consisting of six to eight subunits. Surprisingly, when tested for helicase activity, F540A still showed significant amount of unwinding, with 50–60% of WT (Supplementary Figure S7b and c). As for the G485P/G501P mutant on the a5-linker, EM study revealed that this mutant exclu- sively formed thinner filaments with diameters of 125A ˚ (Figure 2d), 50A ˚ narrower than the 175A ˚ filaments observed in WT MCM. After surveying the WT filaments extensively, we occasionally found very small portion of the large filaments of the WT (about once out of 100 fila- ments) to contain small portion of the 125A ˚ narrow filament (bracketed portion in Figure 2c). Unlike the wide filament that can spiral DNA along its helicalelectro-positivestrip,thenarrowfilamentformedby G485P/G501P mutant would only have sufficient space in the central channel to allow dsDNA to thread straight through the filament, generating little or no untwisting of the bound double helix, which would predict little or no DNA supercoiling to be induced by this mutant MCM protein. Indeed, EM observation of this mutant bound to circular plasmid DNA revealed very little supercoiling (Supplementary Figure S2c). This result is also cor- roborated by the DNA topology footprint assay demon- stratingthatlittlesupercoilingwasinducedbythea5-linker mutant (G485P/G501P) (Supplementary Figure S4a). Testing the biological relevance of a5–a subdomain interactions in vivo We employed yeast genetics to test the biological rele- vance of the a5–a subdomain interactions important for filament formation using yeast Schizosaccharomyces pombe cells. As mentioned previously, the a-subdomain residue F540 at the interface with a5 is highly conserved as F/Y in MCM4 from several eukaryotic organisms (Supplementary Figure S7a), suggesting a conserved role for the equivalent F540 in other organisms in regulating a5interactionsforoligomerization.Previously,weshowed that the F540A ssoMCM mutant could no longer form filament structure, but still retained 50–60% WT helicase activity (Supplementary Figure 7b and c). Therefore, we made the F540A equivalent mutation in S. pombe, which is Y751A on MCM4, to test the phenotype of this mutation in vivo (see ‘Methods’ section). We constructed plasmids containing mcm4 + WT gene or mcm4-Y751A mutant under control of a weakened thiamine-regulated nmt1 + promoter and transformed these into yeast strains with either WT mcm4 + or a temperature-sensitive allele mcm4ts which is not functional at 36 C. Transformants were isolated in the presence of thiamine, which represses the promoter. WT cells express- ing mcm4-Y751A mutant were able to form colonies at all temperatures and at all levels of expression. However, mcm4-Y751A was not able to complement mcm4ts at 36 C, indicating that the mutant MCM4 is not functional (Supplementary Table S2 and Supplementary Figure S8). Interestingly, mcm4-Y751A is toxic in mcm4ts even at permissive temperatures when overproduced (minus- thiamine), as shown by very small colony size or failure toformcolonies,comparedwithWTmcm4 + orthevector control (Supplementary Figure S8). The cells are elongated, indicating a cell cycle delay or arrest. This phenotype indicates that an already attenuated protein (mcm4ts, in this case) is outcompeted by a dominant lethal mutation (mcm4-Y751A) and suggests that the mutant may form non-functional assemblies with cellular MCM. This in vivo result using the equivalent F540A mutant suggests that the a5–a subdomain inter- actions are critical for cell survival. In addition to the mutation of the conserve residue (equivalent of F540) on the a subdomain at the interface witha5discussedabove,wealsowanttoexaminethebio- logical effect of mutating a5inin in yeast. As G485/G501 residuesarepoorlyconservedinthea5linkerregionamong archaeal and eukaryotic MCMs, we generated a mutant by deleting the a5-helix (residues 690–704 of MCM4 from S. pombe) to test the in vivo effect. When the a5-deletion mutant was expressed from the native mcm4 promoter in a plasmid, the mutant was not able to produce colonies followingthetransformationintothetemperature-sensitive strain (mcm4-ts, FY784 strain) under permissive tempera- ture conditions, although the same a5 plasmid was able to successfully produce colonies in the WT strain (FY261 strain) at all three temperatures tested (data not shown). Thisindicatesthatthedeletionmutantexhibitsasynthetic dosagelethalityphenotype. When performing the same transformation experiments withthemutantderivativesexpressedbytheweakestnmt1 (‘81X’) promoter, we were able to successfully obtain colonies following transformation from both mutation types with WT and temperature-sensitive (mcm4 ts) strains under low expression conditions (+thiamine), although the a5-deletion mutant still showed relatively smaller colonies in the mcm4ts host. Upon induction of the promoter (-thiamine), mcm4ts cells expressing either mutant were no longer able to produce colonies, again indicating a toxic phenotype for a5-deletion mutant (Supplementary Figure S8). These in vivo yeast genetics data obtained with the mutants designed to disrupting the a5 and a-domain NucleicAcidsResearch,2013,Vol.41,No.5 3453 interactions for MCM4 of S. pombe, together with the in vitro structural and biochemical data, suggest an important biological role for the interactions between the a5 and a-domain observed in the filament structure. DISCUSSION We described here an unusually wide filament structure of an archaeal MCM that exists both in crystals and solution. The formation of this large, left-handed filament requires the presence of dsDNA. Remarkably, MCM filament formed on DNA is capable of drastically changingDNAtopology.Thistopologychangeisresulted from untwisting the bound dsDNA, changing the local helical parameters of the duplex from B-form to a looser isoform similar to A-form DNA. Furthermore, we identified the structure elements critical for filament formation through in vitro biochemical and EM studies and showed that mutation on these structural elements impacted MCM4 function and cell survival through in vivo genetics study in S. pombe yeast. Onepossiblefunctionalimplicationofthenewstructural and biochemical data described here could be that MCM helical complexes bind and untwist DNA at origins to facilitatestrandseparation.Similarly,bacterialreplication factorDnaAhasbeenreportedto‘screw’dsDNAwithina right-handed helical filament, in a comparable manner to RecA, which is thought to catalyze origin-DNA melting and replication initiation (44). AAA+ family enzymes, including MCM, share a core fold similar to that of the RecA family of proteins and perform similar functions in DNA remodeling. The pitch and angle of RecA filament subunits can adjust in response to ligands such that a change in filament morphology occurs, and RecA and Rad51 (a eukaryotic RecA homolog) form both right- andleft-handedhelicalfilamentsaroundDNAtofacilitate disruption of the duplex (45,46). The role for MCM filaments in replication initiation would fit well with the current understanding of pre-RC architecture. The central channel of this unusually wide filament is large enough to support a replication bubble andallowanMCMringtoclampdownonnewlyexposed ssDNA. This would also allow the filament to protect ssDNA from prolonged exposure to the cellular environ- ment. In this case, the ORC could act as a nucleation point for filament growth (12). A modest conformational change via the a5 (and possibly other structural elements) would allow a transition of MCM from the filament form to the hexamer from that is the active helicase conform- ation.TheinvitroreconstitutedMCMdoublehexameron dsDNA shows no DNA melting (6). One of the possible scenarios is that MCM initially loaded to the pre-RC as a double hexamer would transition to a filament to initiate origin melting, excluding the lagging strand and then closing back into a ring. However, the process in this scenario seems inefficient. Alternatively, the filament and double hexamer may exist together around origins, parti- tioningtheroleofmeltingandunwindingbetweenthetwo structural forms of MCM. In any case, other replication factors are certainly involved in helicase activation. GINS andCdc45arereportedtopromoteaswitchfromanopen ‘lockwasher’ofMCM2-7toaplanarring(47).Givenour data, it is possible that GINS and Cdc45 could also assume a role in switching MCM from a filament to a ring at melted origin DNA. It is noted that in addition to the previous right-handed ‘lock washer’ structure, a left-handed‘lockwasher’formofMCM2-7(witharrange- ment similar to a filament) was recently reported (41). Notably, the MCM proteins that associate with unreplicated chromatin regions away from the origins (the MCM paradox) remain largely uncharacterized. So far, two chromatin-bound MCM populations have been described: an ‘associated’ population that can be removed by high salt, and a ‘loaded’ population that was shown to be origin-DNA-bound hexamers/double hexamers (13,20–23). As the MCM filaments described here could only be formed in relatively low salt concentration, this salt-sensitive nature of the filament form suggests that they may be categorized as the ‘associated’ population. Most studies focus on pre-RC assimilated MCM complexes at replication origins; however, a larger portion of MCM proteins is distributed over a wide range of unreplicated chromatin and distal to replication origins (13). It is an intriguing question whether the filament form of MCM partially accounts for those MCMs associated with chromatin regions away from the origins, as mentioned in the MCM paradox. MCM’scapabilityofalteringDNAtopologysuggestsa possible mechanism for MCM proteins to regulate the diverse array of biological processes in addition to repli- cation, such as chromatin remodeling and transcriptional regulation. Evidence suggests that MCM proteins (likely the origin distal MCM population) are involved in add- itional functions outsidegenome replication, suchas tran- scription, chromatin remodeling and tumor suppression [reviewed by Forsburg (48)]. It is conceivable that the ability of MCM filament formation on DNA to cause dramatic supercoiling of distal dsDNA would likely influ- ence chromosome structure, which should impact the regulation of gene expression and other aspects of DNA metabolism. A more detailed structure/function under- standing paired with further in vivo data will be required to resolve these exciting possibilities. ACCESSION NUMBERS Theatomicmodelreportedherehasbeendepositedtothe Protein Data Bank under the accession number 4FDG. SUPPLEMENTARY DATA Supplementary Data are available at NAR Online: Supplementary Tables 1–3, Supplementary Figures 1–9 and Supplementary Movies 1–2. ACKNOWLEDGEMENTS We thank the staff of USC NanoBiophysics core and the staffatsynchrotronbeamlines23IDand19IDatArgonne National laboratory, and 5.0.2, 8.2.1, 8.3.1 beamlines at 3454 NucleicAcidsResearch,2013,Vol.41,No.5 Berkeley’sALSforassistancewithdatacollection.Weare grateful to Pavel Alfone, Nat Echols of PHENIX group and Axel Brunger for early access to the program DEN (imbedded inCNS 1.3) designedfor low-resolution refine- ment, Lauren Holden and Jared Peace for critical discus- sion and manuscript proofing and Lawrence Lee for helpful discussion. FUNDING Funding for open access charge: NIH [GM080338 and AI055926 to X.S.C., GM071940 to Z.H.Z. and GM GM059321 to S.F.]. Conflict of interest statement. None declared. REFERENCES 1.Aparicio,O.M., Weinstein,D.M. and Bell,S.P. (1997) Components and dynamics of DNA replication complexes in S. cerevisiae: redistribution of MCM proteins and Cdc45p during S phase. Cell, 91, 59–69. 2.Chong,J.P., Mahbubani,H.M., Khoo,C.Y. and Blow,J.J. 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MCM Complexes Form A Filament To Remodel DNA Structure and Topology SUPPLEMENTAL INFORMATION: N-terminal Hairpin Conformation Supports Filament Electrostatic Surface The superimposition of the N-domain of the subunit from the filament structure with the N- ssoMCM (2VL6), which has an r.m.s.d of 1.097, reveals that the N-terminal hairpin (Nt-hp) in MCM filaments adopts a position different from that in the previous N-ssoMCM and N-mtMCM structures, it shifts away from the N-terminal B-subdomain and towards the allosteric communication loop (ACL) (Fig. 4a). This Nt-hp conformational change allows it to make contacts with neighboring B subdomain of the next subunit in the filament formation, which is in contrast to N-domain of the hexamers (2VL6) in which Nt-hps are pointing into the central channel solvent in a planar array. Furthermore, this shift of the Nt-hp that carries positively charged residues on it changes the electro-positive surface layout from the circled ring on a hexamer inner surface to a spiral on the inner filament surface, traveling along the helical filament (Fig. 4b, Fig. 5a-c). The Wide Filaments of WT MCM Shorten the Length of dsDNA If wide filaments are spiraling DNA inside the channel to transform B-DNA to A-DNA, the length of the filament on a 1,000 bp DNA should be significantly shorter than the length in B-DNA (S- Fig. 8a). Likewise, if the narrow filaments of α5-linker mutant bind the DNA straight through the central channel (S-Fig. 8a), their lengths should be near that of a 1,000 bp DNA in B-form. To test this, we measured the length of a population of the WT filaments and the α5-linker mutant filaments formed on a linear 1,000 bp dsDNA (S-Table 3). The average filament length measured for the mutant was 284 nm (with a maximum length of 331 nm), which is near the length calculated for an extended linear B-form DNA (340 nm), indicating that DNA is likely threaded straight through the central channel. Strikingly, the average length of WT filaments was 103 nm with a maximum length of 162 nm. This is far less than the calculated 340 nm for straight B-form DNA (S-Table 3). The lengths are even shorter than the calculated 240 nm for straight A-form DNA could be partially be accounted to the unstable ends of the wide filaments, leaving portions of the dsDNA uncoated at both ends which could not be visualized. Nonetheless, these qualitative observations are consistent with dsDNA being spiraled along the central channel wall in wide filaments, shortening the linear dsDNA, and generating supercoils in circular plasmid DNA (S-Fig. 2a, S-Fig. 4b). On contrary to the WT MCM filament, the narrow filaments of α5-linker mutant is consistent with the straight through binding of the dsDNA, which also explains why this filament does not generate supercoiling in circular plasmid DNA (S-Fig. 2c, S-Fig. 4a). Slaymaker et al. MCM Complexes Form A Filament To Remodel DNA Structure and Topology SUPPLEMENTAL FIGURES S-Table 1 Data collection and refinement statistics *Highest resolution shell is shown in parenthesis. Slaymaker et al. MCM Complexes Form A Filament To Remodel DNA Structure and Topology S-Figure 1. Samples of electron density map sections. (a, b, c) Three sections of 2Fo-Fc map that show the electron density for the main-chain and side-chain features. (d) Validation graphic of the final structure generated from Polygon program using Phenix. Slaymaker et al. MCM Complexes Form A Filament To Remodel DNA Structure and Topology S-Figure 2. Comparison of filament formation and supercoiling of circular plasmid DNA by WT MCM and the α5-linker mutant. (a) WT ssoMCM filament on circular plasmid dsDNA forms the large filament structure (~175Å wide) and induces heavy supercoils. (b) WT ssoMCM forms a filament on a 1,000 bp linear dsDNA, which has a diameter of ~175 Å, and a length around ~120 nm, much shorter than that of a 1,000 bp linear B-form dsDNA (340 nm). (c) α5-linker mutant forms a narrower filament (~125 Å wide) on circular plasmid DNA, which shows little supercoiling compared with WT. The result is consistent with what is shown in S-Fig.4a-c. (d) α5-linker mutant forms a narrow filament (~125 Å wide) on a 1,000 bp linear dsDNA. The length is ~331 nm, close to that of a 1,000 bp B-form DNA, suggesting no significant shortening of the 1000 bp dsDNA, which is also consistent with the lack of supercoiling on circular plasmid DNA for this mutant, as shown in panel-c here. All scale bars (black) are 50 nm. Slaymaker et al. MCM Complexes Form A Filament To Remodel DNA Structure and Topology S-Figure 3. (Related to Figure 5d) The mutations of the positively charged residues on the electro-positive DNA binding strip do not disrupt oligomerisation. (a) Gel filtration profiles of MCM WT and mutants containing mutations on the electro-positive DNA binding strip, showing these mutants behave similarly as WT in oligomerisation. The buffer contains 250 mM NaCl. The molecular marker positions as well as the expected hexamer and monomer peak positions are indicated by arrows. (b) Elution profile of molecular markers on the same column. Slaymaker et al. MCM Complexes Form A Filament To Remodel DNA Structure and Topology S-Figure 4. (Related to Figure 4c) Comparison of DNA conformational changes induced by WT and mutant MCM proteins. (a) DNA topology footprint of α5-linker mutant of MCM (G485P/G501P), showing little supercoiling of the circular plasmid DNA was induced by the mutant MCM when compared to the result using WT MCM in panel-b. This result is consistent with the EM result that shows the thinner filament formed on DNA by this α5-linker mutant does not induce supercoil on circular plasmid (see S-Fig. 2c). (b) WT MCM topology footprint, showing heavily supercoiled DNA conformation induced by WT MCM, consistent with the EM study shown in S-Fig. 2a. (c) WT MCM topology footprint assay as in panel-b, except that chloroquine was added to the agrose gel during electrophoresis analysis to verify the extend of negative supercoils induced by MCM, as chloroquine is known to shift negatively coiled circular DNA towards the sample wells relative to chloroquine free gels shown in panel-b. R: Relaxed plasmid generated from nicked negatively supercoiled pBR233 plasmid by topoisomerase I treatment, and lanes 1-7 in all panels: the relaxed plasmid was incubated with increasing concentrations of MCM (1-10 µM of MCM proteins). OC = open circle, (-) SC = negative supercoiling. Lk = linker number. Slaymaker et al. MCM Complexes Form A Filament To Remodel DNA Structure and Topology S-Figure 5. The structure of two neighboring ssoMCM subunits in the filament, showing the conserved interface on one subunit (drawn in surface structure) for binding α5 from a neighbor subunit (drawn in ribbon). The degree of conservation on the surface structure is obtained from sequence alignment of ssoMCM with S. pombe, H. sapiens, S. cerevisiae, and X. laevis MCM proteins 2-7, and shown by Consurf server coloring scheme (at the bottom). Red is highly conserved between MCMs and blue is highly variable. Helix-5 (α5) (indicated) docks to a highly conserved region (colored in red) of the three helices of the α-subdomain that contains the highly conserved residue F540 (see S-Fig. 7a). Slaymaker et al. MCM Complexes Form A Filament To Remodel DNA Structure and Topology S-Figure 6. Gel filtration (Superose-6) chromatography assay of MCM mutants, with molecular marker elution profile shown in panel-c at the bottom. (a) Gel filtration profiles of ssoMCM WT and mutants containing G485P/G501P mutations on the α5 linker and F540A at the interface between α5 and α-domain of two neighboring subunits. The results reveal that the two mutants behave differently from WT in oligomerization. WT profile (indicated by *) has a peak at the hexamer position with molecular weight of approximately 480 kD. The mutants eluted at peak positions of approximately 210 kD (around the molecular weight of a trimer), indicating weakened inter-subunit interactions for the mutants at the assay condition. The gel filtration chromatography was on Superose-6 analytical column in a buffer containing 20 mM HEPES pH 7.5, 2 mM DTT, and 250 mM NaCl. The molecular marker positions as well as the expected hexamer and monomer peak positions are indicated by arrows. (b) Elution profile of molecular markers. Slaymaker et al. MCM Complexes Form A Filament To Remodel DNA Structure and Topology S-Figure 7. Helicase activity of ssoMCM-F540A mutant at the interface between helix α5 and α- domain. (a) Alignment of S. solfataricus MCM and S. pombe, H. sapiens, S. cerevisiae, X. laevis and D. melanogaster MCM4 proteins around the F540 region one of the three α-domain helices ( α7) which is at the interface with helix α5. The residue aligning to ssoMCM F540 is marked by (*). (b) F540A mutant that can no longer form filament still showed helicase activity, which is ATP dependent. B: boiled fork DNA; UB: unboilded fork DNA without protein, negative control for unwinding; WT: wild type protein; M: F540A mutant protein. The positions of dsDNA and ssDNA are indicated. (c) Helicase activity of ssoMCM-F540A at a range of tested protein concentrations, showing that F540A mutant retained around 50-60% of the WT helicase over a range of protein concentrations. Error bars were derived from 3 separate experiments. Slaymaker et al. MCM Complexes Form A Filament To Remodel DNA Structure and Topology S-Table 2. Complementation data at permissive temperature 32 0 C in S. pombe. The wt and mutant MCM4 expressed in the plasmids is driven under an attenuated nmt promoter that is only induced in the absence of thiamine. Plasmid Wild Type + thiamine Wild Type - thiamine mcm4ts L238P + thiamine mcm4ts L238P - thiamine pNR29 (mcm4 + ) + + + + pSLF372 (vector) + + + + pNR30 mcm4- Y751A-HA + + + +/- pNR31mcm4- Δ690-704 ( Δ ) + + - - S-Figure 8. Phenotypes of mutants (mcm4-Y751A) and wild type mcm4 (mcm4 + ) plasmids transformed in the mcm4 temperature sensitive and in wild type strains. Candidates were streaked on EMM-URA+ thiamine and - thiamine plates and incubated at 25ºC, 32ºC and 36ºC for 7 days. The representation is following 5 days of incubation. Presence of thiamine suppresses the expression WT or mutant MCM protein from the transformed plasmid. The drawing on the bottom indicates the layout of the six plates and the six experiments on each plate with particular clones and the cellular mcm4 genetic backgrounds. Slaymaker et al. MCM Complexes Form A Filament To Remodel DNA Structure and Topology S-Table 3. A survey of the filament lengths of WT MCM and α5-linker mutant assembled on 1,000 bp dsDNA, showing significant differences (also see S-Fig.3). A fully extended 1,000 bp linear dsDNA is 340 nm in B-form, and is 240 nm in A-form. Wild type α5-linker mutant Image # Length (nm) Image # Length (nm) 101 140.35 34 276.46 99 79.57 32 294.68 97 89.84 29 295.85 93 77.42 27 319.48 86 121.27 25 268.36 84 99.42 23 202.15 80 96.69 18 309.59 78 151.75 16 310.26 76 162.45 14 261.49 56 100.59 12 303.48 54-1 121.21 10 331.90 54-2 111.71 3 301.71 46 85.77 2 211.65 33 93.02 29 60.62 25 84.40 17 65.77 13 120.60 Average Length 103.47 Average length 283.62 Slaymaker et al. MCM Complexes Form A Filament To Remodel DNA Structure and Topology S-Figure 9. Models illustrating how dsDNA (red) binds inside the wide and narrow filaments. (a) Model showing how dsDNA binds to the electro-positive surface inside the 175Å wide filament in a helical manner. This binding mode is consistent with the significantly shortened linear dsDNA length, the heavy supercoiling of circular plasmid, and the electro-positive strip spiraling along the inner surface of the filament. (b) Model showing how dsDNA threads through the 125Å narrow filament in a straight manner. This binding mode is consistent with the lack of ability to shorten linear dsDNA length, inability to generate supercoiling on circular plasmid. 148 Appendix A Identifying potential Cds1 phosphorylation sites on mcm4+: A1. Purpose: Mcm4 was shown to associated with Cds1 and undergo a Cds1 dependent phosphoryaltion (Bailis et.al 2008). This indicated that Mcm4 is a direct substrate of Cds1. Further it was shown in the same work that mcm4-M86 (mcm4ts) when pre incubated in hydroxyurea (HU) causes the activation of Cds1. The activation of the checkpoint is sufficient to suppress the lesions that are associated with mcm4ts. The data suggested that both mcm4+ and mcm4ts are substrates of Cds1. Through personal communication from another lab that performed mass spectrometry, there were several residues that were identified as undergoing HU-specific phosphorylation. But the residues that were identified did not match the putative Cds1 consensus sequence(S/T-P-X- K/R). The residues identified through mass spectroscopy were on location S30, S38, S81, ST95 on mcm4. We were interested in making point mutations on these residues by changing the S/T to Alanine ; and to create a combination mutant with all 4 residues mutated to Alanine. By creating the mutations were wanted to know: 1) If the mutations were able to complement the mcm4ts (mcm4 M-68) and mcm4∆ in a diploid. 2) If the mutations are able to abolish the phosphorylation that is observed with HU treatment. 149 A2. Methods: Site directed mutagenesis and cloning: Phusion site directed mutagenesis kit # F-541 ( Thermo Scientific) was used to create the specific point mutations according to the manufacturers protocol. Following the mutation generation, the mcm4 containing the point mutants were cloned to puR19N (plasmid # 101 in lab collection) to perform complementation assays. Following a successful complementation the mutation was integrated to the genome to create a stable strain for experiments (mcm4-4SA) ( FY 5251, FY 5264, FY 5255) Generated plasmids, mutation location and primers are listed in Table A.1. The entire mcm4+ was sequenced to ensure that no additional mutations were introduced during PCR. Once the mutations were intergrated in to the genome selected genomic DNA was extracted from the strain and was PCR amplified and sequenced to ensure that the strain carried the 4 point mutations. Complementation assay: Complementation assay using the temperature sensitive mcm4ts (FY784) , wild type (FY 528 or FY261), and deletion mcm4∆ (FY 857) using a dipoloid was performed as previously described in (Gómez et.al 2002). 150 Serial dilutions and viability: Cells were grown in YES to an OD of 0.5 and were counted and diluted to ensure that all strains used had equal number of cells. Fivefold serial dilutions were next plated on different concentrations of HU and MMS to test for sensitivity. Early log phase cells (OD. 0.3) growing in liquid media were exposed to 12Mm HU and allowed to grow at 25°C for 8hrs and plated on YES at 25°C for viability (Figure A.1.a and A.1.b). Strains generated for these experiments are listed in Table A.3. Mobility shift: Cells were grown in 25°C either treated or untreated with 12mM hydroxyl urea for 4hrs. Cells were next treated with10X stop buffer containing 2% Sodium Azide and were incubated on ice for 10mins before harvesting the cells. Cells were subsequently washed twice with 1X PBS and whole cell proteins were extracted using trichloric acid (TCA) (Foiani et.al 1994). The extractions were quantified using Pierce BCA Kit. Equal amounts of -100ug of protein was loaded on 8% SDS PAGE gels and blotted using primary antibodies 16B12 anti HA Covance in 1:1500 dilution overnight at 4C. After washing with PBST it was blotted with anti-mouse-IgG- HRP (1:5000 sigma). 1:1500 PCNA (Santa Cruz) was used as the loading control. 151 A3. Results: The result of the complementation assay is summarized in Table A.2. The results showed that single mutations (S30A, S38A,S81A,T95A) and the combination mutation were able to complement both mcm4ts and mcm4 ∆. We didn’t see a difference in viability by either chronic or acute exposure to HU or MMS. The cells showed a similar viability to wild type as seen in Figure A.1. We also didn’t see a notable abolishment of the phosphoshift in the strain containing the four point mutations (S30A,S38A,S81A,T95A). It was not convincing to state that the mutations were able to abolish the phophoryaltion caused by HU treatment figure A.2. We saw that the double mutant with cds1∆ was sensitive to HU. Table A.1: Plasmids constructed and used for this work: Plasmid Name Collection # Mutation Gene Primers Used pNR-A 1498 S30A mcm4 1165F, 1166R pNR-C 1500 S38A mcm4 1167F, 1168R pNR-D 1501 S81A mcm4 1169F,1170R pNR-E 1504 T95A mcm4 1171F,1172R pNR9 1589 S30A,S38A,S81A,T95A mcm4 1429F and 1430R(30,38) 1447F and 1448R(81,95) 152 Table A.2: Complementation results: Table A.3: Stains used for this study: Strain # Genotype Source FY 261 h+ can1-1 leu1-32 ade6-M216 ura4-D18 Our stock FY 528 h+ his3-D1 ade6-M210 ura4-D18 leu1 Our stock FY 784 h+ cdc21-M68 ura4-D18 leu1-32 ade6-M210 can1-1 Our stock FY 857 h-/h+ ∆cdc21::his3+/+ ura4-D18/ura4-D18 leu1-32/leu1-32 ade6-M210/ade6-M216 his3-D1/his3-D1 (mcm4) Our stock FY 5251 h-cdc21::pNR13[S30A,S38A,S81A,T95A] his3-D1 ade6- M216 leu1-32 ura4-D18 This work FY 5264 h+cdc21::pNR13[S30A,S38A,S81A,T95A] ade6-M216 leu1- 32 ura4-D18 This work FY 5265 h-cdc21::pNR13[S30A,S38A,S81A,T95A] ade6-M216 leu1- 32 ura4-D18 This work FY 5266 h-cdc21::pNR13[S30A,S38A,S81A,T95A]ade6-M210 leu1-32 ura4-D18 This work FY 5490 h- cdc21::pNR13[S30A,S38A,S81A,T95A] cds1::ura4+ his3-D1 ade6-M216 leu1-32 This work Plasmid Name Mutation Complementation with mcm4ts Complementation with mcm4 ∆ pNR-A S30A YES YES pNR-C S38A YES YES pNR-D S81A YES YES pNR-E T95A YES YES pNR9 S30A,S38A,S81A,T95A YES YES 153 FY 5491 h+ cdc21::pNR13[S30A,S38A,S81A,T95A] cds1::ura4+ ade6-M216 lue1-32 This work FY 5686 h-cdc21*-HA pNR13[S30A,S38A,S81A,T95A] his3-D1 ade6- M216 leu1-32 ura4-D18 This work FY 5687 h+cdc21*-HA pNR13[S30A,S38A,S81A,T95A] his3-D1 ade6-M216 leu1-32 ura4-D18 This work Figure A.1a: mcm4-4SA containing the 4 point mutants doesn’t show HU, MMS or temperature sensitivity 154 Figure A.1.b.Relative viability in 12mM HU. Figure A.2 Western blot to look at Hydroxyurea specific phosphorylation A4. Conclusion: The results from this work show that although these residues were identified as getting phosphorylated by HU, after mutating the residues the cells were not convincingly abolishing phosphorylation compared to wild type in response to HU treatment. Even if does have an effect on the phosphorylation, there are many Cds1 target sites on the protein that would still be 155 contributing towards the phosphorylation and mutating many more of those sites will might be needed to see a definitive effect. A5. Appendix A References: Bailis, J. M., Luche, D. D., Hunter, T., & Forsburg, S. L. (2008). Minichromosome maintenance proteins interact with checkpoint and recombination proteins to promote s-phase genome stability. Mol. Cell. Biol. 28: 1724–38. Foiani, M., F. Marini, D. Gamba, G. Lucchini, and P. Plevani. 1994. The B subunit of the DNA polymerase alpha-primase complex in Saccharomyces cerevisiae executes an essential function at the initial stage of DNA replica- tion. Mol. Cell. Biol. 14:923–933 Gómez, E. B., Catlett, M. G., & Forsburg, S. L. (2002). Different phenotypes in vivo are associated with ATPase motif mutations in Schizosaccharomyces pombe minichromosome maintenance proteins. Genetics, 160(4), 1305–18. 156 Appendix B Characterizing other mcm4 mutants B1.Purpose: The N terminus region of mcm4 has been studied in detail in budding yeast and fission yeast (Sheu & Stillman, 2010,Sheu et al.,2006,Sheu et al.,2014,Masai et al., 2006). It was shown in budding yeast that deletion of certain regions of the N terminus had different effects in the origin firing and fork progression. The mcm4 N terminal 74-174 region was shown to have an intrinsic inhibitory role in origin firing, and the 2-145 region was shown to play a role in S phase progression. The mcm4 ∆74-174 is able to bypass DDK regulation (Sheu & Stillman, 2010). We were interested to test certain truncations studied in the budding yeast system in the S.pombe system to evaluate if they show similar cell cycle dynamics .We made the same deletions in pombe in mcm4 ; ∆2-73, ∆2-133 and ∆72-153 and performed complementation assays. In addition to the N terminal region a C terminal truncation deleting amino acid 768-887 was created to remove the entire C terminus region. B2.Methods: Site directed mutagenesis and cloning: Phusion site directed mutagenesis kit # F-541 (Thermo Scientific) was used to create the specific mutations. Following the mutation generation the plasmids were cloned to puR19N to perform complementation assays and were integrated to the genome to create a stable strain for experiments. Generated plasmids, mutation location and primers are listed in Table B.1. The 157 entire mcm4+ was sequenced to ensure that no additional mutations were introduced during the process of the PCR. Genomic DNA was extracted from the strains created and was PCRed and sequenced to ensure that the mutations were still present in the strain. Complementation assay: Complementation assay using temperature sensitive mcm4ts (FY784) and wild type (FY 528 or FY261), and mcm4∆ using a diploid (FY 857) was carried out as previously described in (Gómez et.al 2002). Results are recorded in Table B.1. Serial dilutions and viability: Cells were grown in YES to an OD of 0.5 and were counted and diluted to ensure that all strains used for the assay had equal number of cells. Fivefold serial dilutions were plated on different concentrations of HU and MMS to test for any sensitivity. Early log phase growing cells (OD. 0.3) were exposed to 12mM HU and allowed to grow at 25C for 8hrs and plated on YES at 25C to check for viability. (Figure B.1 and B.2). Strains used for the assays are listed in Table B.2. B3.Results: Although all three mutations were able to complement mcm4ts and mcm4∆ we were only able to integrate 2-73 mutation into the genome to perform experiments. The C-terminal truncation; 768-887∆ was unable to complement mcm4ts. No further experiments were performed with this mutant. 158 Figure B.1 : Viability in HU treatment Figure B.2 Sensitivity to MMS and HU 159 Table B.1: List of plasmids created for this work and complementation results: Table B.2: Stains used for this study: Plasmid Name Collection# mutation location on mcm4+ primers Complementation with mcm4ts Complementation with ∆mcm4 pNR18 1638 2-73 1697F,1698R YES YES pNR20 1640 2-133 1698 F,1699R YES YES pNR51 1895 72-153 1757F, 1758R YES YES pNR44 1887 768-887 2439F,2440 R NO ND Strain # Genotype Source FY 261 h+ can1-1 leu1-32 ade6-M216 ura4-D18 Our stock FY 528 h+ his3-D1 ade6-M210 ura4-D18 leu1 Our stock FY 784 h+ cdc21-M68 ura4-D18 leu1-32 ade6-M210 can1-1 Our stock FY 857 h-/h+ ∆cdc21::his3+/+ ura4-D18/ura4-D18 leu1-32/leu1-32 ade6-M210/ade6-M216 his3- D1/his3-D1 (mcm4) Our stock FY 5688 h-cdc21::pNR22[ deletion2-73] his3-D1 ade6- M216 leu1-32 ura4-D18 This work FY 6360 h- cdc21::pNR22[ deletion2-73] cds1::ura4+ ade6-M216 leu1-32 ura4-D18(mcm4) This work FY 6361 h+cdc21::pNR22[ deletion2-73] cds1::ura4+ his3 -D1 ade6-M210 leu1-32 ura4-D18(mcm4) This work 160 B4.Conclusion: We saw that the N terminal truncation mutations were able to complement mcm4-M68 and mcm4∆ diploid and there was no apparent sensitivity to HU and MMS in the deletion mutant. When combined the 2-73 truncation with cds1∆ we saw that the double mutant was more sensitive to hydroxyurea than the 2-73∆ parent strain. No further studies were carried out with the mutant strain since there were no interesting phenotypes to follow. The C terminal truncation mutant was unable to complement the mcm4-M68. No further experiments were performed with these mutants. B5. Appendix B References: Gómez, E. B., Catlett, M. G., & Forsburg, S. L. (2002). Different phenotypes in vivo are associated with ATPase motif mutations in Schizosaccharomyces pombe minichromosome maintenance proteins. Genetics, 160(4), 1305–18. Masai, H., C. Taniyama, K. Ogino, E. Matsui, N. Kakusho et al., 2006 Phosphorylation of MCM4 by Cdc7 kinase facilitates its interaction with Cdc45 on the chromatin. J. Biol. Chem. 281: 39249–61. Sheu, Y.-J., Kinney, J. B., Lengronne, A., Pasero, P., & Stillman, B. (2014). Domain within the helicase subunit Mcm4 integrates multiple kinase signals to control DNA replication initiation and fork progression. Proceedings of the National Academy of Sciences of the United States of America, 111(18), E1899–908. 161 Sheu, Y.-J., & Stillman, B. (2006). Cdc7-Dbf4 phosphorylates MCM proteins via a docking site- mediated mechanism to promote S phase progression. Molecular Cell, 24(1), 101–13. Sheu, Y.-J., & Stillman, B. (2010). The Dbf4-Cdc7 kinase promotes S phase by alleviating an inhibitory activity in Mcm4. Nature 463: 113–117 162 Appendix C Yeast Two Hybrid (Y2H) to test if the C terminus of mcm4 was responsible in interacting with mcl1 C1. Purpose: Fork protection complex that consist of Swi1, Swi3 and Mrc1 helps in maintaining a stable replisome by forming a connection between the helicase and polymerases. Swi1 and swi3 are thought to be linked with the leading strand while mcl1 is thought to be linked with the lagging strand. Further it has been shown that swi1, swi3 and mcl1 all show sensitivity to MMS (Sommariva et al., 2005, Williams & McIntosh, 2002) . As described in chapter 3; mcm4c106 relies on the swi1, swi3, and mrc1 for viability. But this lethality is not observed with mcl1-1. It has been shown that GINS and Ctf4 [components of the RPC] is crucial to couple MCM2-7 to DNA polymerase α as shown in figure C.1 (Gambus et al. 2009). Studies of Ctf4/Sp mcl1 has shown that Ctf4 binds directly to the GINS subunit of the CMG helicase and to the catalytic subunit of Pol α via the C- terminal half of the protein (Gambus et al. 2009), and interact with cdc45 via its C terminus region. We speculated that mcm4c106 might be interacting with mcl1 via its C terminus and when deleting the C terminus it would abolish this interaction resulting in the cells to be relying on Swi1 and Swi3 for viability. We performed a yeast two hybrid to address the question as to if there is a direct interaction that is observed between mcl1 and mcm4c106. 163 Figure C.1:.Hypothesis of mcm4 C terminus interacting with mcl1 C2. Method: To test this hypothesis, I performed Yeast two hybrid assay, as shown in figure C.2. The Following constructs were made and used for the experiments. a) Mcl1 full length and Mcm4 Full length protein cloned as prey and bait b) Mcl1 full length and mcm4 protein lacking 106 amino acids from the C terminus cloned as prey and bait c) Mcl1 full length and only 106 amino acids of mcm4 protein clones as prey and bait The diagram of the constructs is shown in figure C.3. Figure C.2 Y2H system. Adapted from (Stephens and Banting 2000) 164 Figure C.3. Mutants tested in the Y2H assay. Clontech Matchmaker GAL4 Two Hybrid Sytem 3 was used for the Y2H assays. AH109 yeast strain with Ade2,His3, lacz under the control of distinct GAL4 was used to test for the interactions. Cloning vectors for the assay is shown in table C.1 followed by the plasmids in constructed for the assay in Table C.2. The mcm4 gene was PCRed from plasmid 466 [pTZ18R- cdc21] introducing appropriate restriction site for cloning using the defined primers. Table C.1: cloning vectors for Y2H Vector Fusion Epitope Yeast Selection Bacterial selection Collection # pGBKT7 DNA/Bait C-myc TRP1 Kan 1491 pGADT7 AD HA LEU2 Amp 1527 165 Table C.2: Y2H plasmids created for the experiments: Plasmid Protein Yeast Selection Vector plasmid Collection number Primers reference pNR41 Full mcm4 TRP1 pGBKT7 1878 2413F,2415R This work pNR42 Mcm4 lacking 106 amino acids from C terminus TRP1 pGBKT7 1879 2413F,2414R This work pNR39 C016 from mcm4 TRP1 pGBKT7 1862 2370F,2371R This work pNR56 Mcl1 LEU2 pGADT7 1905 2400F,2401R (pcr mcl1 from cDNA) This work pNR43 Deltion 690-704 TRP1 pGBKT7 1886 2413F,2415R for cloning; Primers 1738F and 1739 was used to created the 690-704 deletion. This work pNR52 72-153 Deletion TRP1 pGBKT7 1896 1757F,1758 R to create truncation This work pNR46 Rad22 LEU2 pGADT7 1889 2445F, 2446R to PCR out the rad22+ This work pNR45 Mst1 TRP pGBKT7 1888 2442F,2443R to PCR out mst1+ This work pNR50 2-73 TRP pGBKT7 1894 2461F and This work 166 C3. Results: The results that were observed are summarized in table C.3. In summary we didn’t observe an interaction with mcm4 and Ctf4/mcl1 based on the Y2H candidate approach. The controls for the testing system were functional, as shown by the growth on the medium stringent plates for the positive control and no growth on the same plates for the negative control as shown in figure C.3. truncations 2415R was used for cloning. 1697F and 1698 to create truncation pGBKT7- pob3#1 Pob3 TRP pGBKT7 1522 Our stock Tabancay (unpublished) pGADT7- spt16#1 Spt16 LEU pGADT7 1523 Our stock Tabancay (unpublished) 167 Table C.3: Y2H interaction results: -Leu, -Trp (lowest stringency) Leu,-Trp,- His(Medium stringency) -Leu, -Trp,- Ade(Medium stringency) Pob3+Spt16 Positive control +++ ++ ++ pGBKT7 ; pGADT7 Negative control ++ No No Full length [pNR41]mcm4+pGADT7(v) Control ++ NO NO Full length Mcl1[pNR56] + pGBKT7 (v) Control ++ NO NO C106 [pNR39]+ pGADT7(v) Control ++ NO NO Truncated mcm4[pNR42]+pGADT7 Control ++ NO NO Full mcm4[pNR41] +mcl1 Experimental ++ NO NO [pNR39]+mcl1[pNR56] Experimental ++ NO NO [pNR42]+mcl1[pNR56] Experimental ++ NO NO 168 Figure C.4: Controls for Y2H growth seen on lowest and medium stringency plates supplemented with 3AT to reduce non specific interactions. C4. Conclusion: Based on the Y2H data we were not successful in showing that mcl1 and mcm4 had a physical interaction. This doesn’t mean to say that mcm4 and mcl1 might not have an interaction in an indirect fashion. A cDNA library screen could be done using the truncated region as the bait to identify any candidates that could potentially link the mcm4 to the association of the replisome. C5. Appendix C References: Gambus, A., van Deursen, F., Polychronopoulos, D., Foltman, M., Jones, R. C., Edmondson, R. D., Labib, K. (2009). A key role for Ctf4 in coupling the MCM2-7 helicase to DNA polymerase alpha within the eukaryotic replisome. The EMBO Journal, 28(19), 2992–3004. Lou, H., Komata, M., Katou, Y., Guan, Z., Reis, C. C., Budd, M., Campbell, J. L. (2008). Mrc1 and DNA polymerase epsilon function together in linking DNA replication and the S phase checkpoint. Molecular Cell, 32(1), 106–17. Stephens, D. J., & Banting, G. (2000). The use of yeast two-hybrid screens in studies of protein:protein interactions involved in trafficking. Traffic (Copenhagen, Denmark), 1, 763–768. 169 Appendix D Determine strand specificity during replication using APOBEC3G D1. Purpose: Given that mcm4c106 was relying on the fork protection complex components for viability we hypothesized that mcm4 is linked with the FPC via its C terminus. Therefore removing the C terminus would abolish the interaction which would cause a collapse in the fork resulting in the excessive formation of ssDNA, as seen with the CFP (Rad11foci). We wanted to see if mcm4c106 had strand specificity during the DNA replication process. To test this idea we used a method in which a known point mutation was generated using a mutagenesis system and based on the signature of the mutation to determine the strand that is exposed during the replication process. To perform the experiment we used an APOBEC3G gene containing plasmid. Similar methods have been used in budding yeast, and fission yeast systems to define the polymerases δ, and ε to the leading and lagging strand (Nick McElhinny et al., 2008, Miyabe et.al., 2011). The well established idea is that the division of labor between the two strands are assigned to polδ (as the lagging strand polymerase) and polε (as the leading strand polymerase) (Kunkel & Burgers, 2008,Nick McElhinny et al., 2008, Miyabe et.al., 2011,Miyabe et al., 2015, Johnson et.al, 2015). But more recent work by Johnson et.al show that pol δ is responsible in generating both strands and that the pol ε proof reading was essential to remove any of the errors that were incorporated by pol δ during the process of replication (Johnson et.al., 2015). 170 APOBEC (apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like) is a cytidine deaminases that function in innate immunity (Smith et.al., 2012). APOBEC is known to create specific mutations in which it converts cytosine bases (C) to uracil (U). The C U mutations that are caused during the deamination process results in C T or C G mutations. We exploited this specific mutation pattern generated by APOBEC to determine the exposed strand during replication. Figure D.1: signature of mutation created by APOBEC3G. (Adapted from Roberts et al., 2013) D2.Methods: APOBEC3G gene was PCRed from plasmid 1892 [APOBEC3G; original plasmid was obtained from Dr. Myron Goodman lab] using primers 2472F and 2473R. Plasmid pNR53 (1897) was created, by cloning the APOBEC3G gene into pSGP73 (529) which was under the nmt promoter 171 from REP3X with a N terminal HA tag. The plasmid was sequenced to make sure that no additional mutations were present. The plasmid contained a LEU marker for ease of selection. A URA version of the APOBEC3G plasmid is listed as pNR49 (1893). pSGP73 and pNR53 were transformed to wild type URA 4/5 forward and reverse strains obtained from Tony Carr lab (Miyabe et al. 2011) (FY 7055, FY 7801), and to mcm4c106 URA4/5 forward and reverse strains ( FY 7795 and FY 7648). Figure D.2 indicate the strains used for this experiment. The transformants were selected on plates lacking leucine supplemented with thiamine in a condition that the promoter was turned off. Single transformants were picked and grown at 30°C with thiamine in the media and was washed twice with EMM minus nitrogen. Cells were re- suspended in media lacking leucine and thiamine at approximately 600cells/ul and were allowed to grow for 18-20hrs at 25°C. The media didn’t contain any leucine to ensure that the plasmid was still maintained. The cells were allowed to grow at 25°C for 18hrs at this point the cells were in mid log pahse. In order to generate ssDNA, cells were treated with 12 Mm hydroxyl urea for 4-5hrs or left untreated as the control. Cells were counted after the treatment and 8X10^4 cells were plated on 5FOA phloxen B plates. Plates were allowed to grow at 30°C for 8 days before positive colonies on FOA plates were re-streaked on YES and replica plate on plates lacking uracil and FOA separately to ensure the positive candidates. 1000 cells were plated on YES to determine the number of viable cells after the treatment. Once confirmed; cells were grown in liquid media and genomic DNA was extracted using phenol chloroform methods and PCR was performed using primers 2515F and 2510R to PCR out the URA4 and URA5. Samples were sequenced using 172 primers 2509F,2513F and 2510R. The sequence was aligned against the Ura4 Ura5 sequence and analyzed any mutations. cds1∆ was used as a control. Table D.1 shows the expected mutation signature based on the strand that was exposed during the replication process. Figure D.2. Schematic of the constructs used for the experiment (Adapted from Miyabe et.al 2011) In the forward strain the fork is approaching from the ATG direction where as in the reverse strain the fork is approaching from the TAA direction. Therefore in the forward strain the strand getting synthesized would be the lagging strand while in the reverse strain the strand that is getting synthesized will be the leading strand. Table D.1 expected mutation patter based on the strand that was exposed Reverse Strain Forward Strain Coding Strand Coding Strand C  T ( leading Strand) C T (lagging Strand) G  A ( lagging Strand) G A (leading Strand) 173 Results: Figure: D.3 Growth of APOBEC3G plasmid transformed to Fwd ura4:ura5AH(AseI/HindIII) ura5ÆAH::hyg (Wild Type) and ura4:ura5AH(AseI/HindIII) ura5ÆAH::hyg cdc21-c106::kan It is seen that the when APOBEC3G is transformed to mcm4c106 under no thiamine condition when the promoter is turned on there is less growth observed with the mcm4c106 mutants compared to when APOBEC3G is transformed to the wild type strain. 174 Strain URA5 URA4 Location Strand FY 7795 X G-->A 2404 lagging Reverse Strain mcm4-c106 X G-->A 2656 Lagging X G-->A 2404 Lagging X G-->A 2245 Lagging X G-->A 1898 Lagging X G-->A 2245 Lagging X G-->A 2058 Lagging X G-->A 2058 Lagging G-->A X 928 lagging G-->A X 928 lagging G-->A X 844 lagging X C->T 2491 Leading X C-->T 2490 leading X C-->T 2491 leading X C-->T 2491 leading X G-->C 2245 lagging X G-->C 2310 lagging G-->C X 672 lagging X G--C 2310 lagging X G-->C 2245 lagging X G-->C 2405 laggng X G-->C 2245 lagging X G-->C 2245 lagging Strain URA5 URA4 Location Strand FY 7648 C--G 2491 Forward Strain mcm4- c106 X G-->A 2245 Leading X G--->A 2245 Leading X G--A 2656 Leading X G--A 1930 leading X G-A 2245 leading X G-A 2245 leading Table D.2. Mutation location on untreated mcm4c106 forward and reverse strains 175 Strain URA5 URA4 Location Strand FY 7795 G-C G-A 672 lagging Reverse Strain mcm4-c106 G--A x 754 lagging G-->A 2245 lagging HU treated G-->A 2058 lagging G-->A 2245 lagging G-A 2245 lagging G-A 1898 lagging G-A 2245 lagging G-A 2245 lagging G-A G-C 845/2245 Lagging G-C/G-A 2321/2405 lagging G-->A 2245 lagging G-->A 2245 lagging G-A 2245 lagging G-C 672 lagging X G-->C 2652 lagging G-C 1898 lagging G-C 580 lagging X G--C 2656 lagging X G-C 2245 lagging X G-->C 2245 lagging X C-T 2491 leading C-G 662 leading C-T 748 leading X C-->T 2112 leading C--T 748 leading Table D.3. Mutation location on HU treated mcm4c106 forward and reverse strains 176 Strain URA5 URA4 Location Strand X G--A 2245 leading FY 7648 X G--A 2245 leading Fwd X G--A 2245 leading mcm4c106 X G--A 2245 leading X G--A 2245 leading HU treated X G--A 2245 leading G_-A 844 leading G-C G-A 2495 leading X G-A 2495 leading X G--A 843 leading X G--A 2245 leading X G--A 2245 leading X G--A 2245 leading X G--A 2245 leading X G-C 2493 leading X C-->T 922 lagging X C--T 922 lagging X C--T lagging X C--T 2491 lagging X C--T 2102 lagging X C--T 2491 lagging X C--T 2103 lagging X C--T 2491 lagging After treating cells with HU for 4hrs the cells were plated on 5FOA until colonies were visible. Cells that were growing on FOA would have a mutation in the URA gene that would allow its growth on FOA. Upon genomic DNA extraction and sequencing we didn’t see a bias toward one strand. We saw that in the treated and the untreated conditions, when using the forward yeast strain it was mapping to the leading strand more frequently while when the reverse strain was used it was mapping to the lagging strand. A cds1∆ (forward and reverse) and the wild type strains from the original study were used as controls. These ended up giving similar results. 177 Strain Genotype Source FY 7055 h+ reverse.ura4:ura5AH(AseI/HindIII) ura5ÆAH::hyg ade6- 704 leu1-32 Tony Carr (Miyabe et al. 2011) FY 7801 h+forward.ura4:ura5AH(AseI/HindIII) ura5ÆAH::hyg ade6-704 leu1-32 This study FY 7795 h+ reverse.ura4:ura5AH(AseI/HindIII) ura5ÆAH::hyg cdc21- c106::kan his 3 D-1 ade6-704 leu1-32 This study FY 7648 h+Forward ura4:ura5AH(AseI/HindIII) ura5ÆAH::hyg cdc21- c106::kan ade6-704 leu1-32 his3 ade10 This study FY 7815 h- reverse.ura4:ura5AH(AseI/HindIII) ura5ÆAH::hyg Æcds1::kanMX6-Bioneer ade6-704 leu1-32 This study FY 7816 h+ reverse.ura4:ura5AH(AseI/HindIII) ura5ÆAH::hyg Æcds1::kanMX6-Bioneer ade6-704 leu1-32 This study FY 7817 h-reverse.ura4:ura5AH(AseI/HindIII) ura5ÆAH::hyg mrc1::kanMX6-Bioneer ade? leu1-32 This study FY 7827 h+forward.ura4:ura5AH(AseI/HindIII) ura5ÆAH::hyg Æmrc1::kanMX6-Bioneer leu1-32 ade6-704 This study FY 7828 h-forward.ura4:ura5AH(AseI/HindIII) ura5ÆAH::hyg Æmrc1::kanMX6-Bioneer leu1-32 ade6-704 This study FY 7829 h-forward.ura4:ura5AH(AseI/HindIII) ura5ÆAH::hyg Æcds1::kanMX6-Bioneer leu1-32 ade6-704 This study FY 7830 h+forward.ura4:ura5AH(AseI/HindIII) ura5ÆAH::hyg Æcds1::kanMX6-Bioneer leu1-32 ade6-704 This study Table D.4: Strains used for this study: 178 Appendix Reference: Bailis, J. M., D. D. Luche, T. Hunter, and S. L. Forsburg, 2008 Minichromosome maintenance proteins interact with checkpoint and recombination proteins to promote s-phase genome stability. Mol. Cell. Biol. 28: 1724–38.. Gambus, A., F. van Deursen, D. Polychronopoulos, M. Foltman, R. C. Jones et al., 2009 A key role for Ctf4 in coupling the MCM2-7 helicase to DNA polymerase alpha within the eukaryotic replisome. EMBO J. 28: 2992–3004. Gómez, E. B., M. G. Catlett, and S. L. Forsburg, 2002 Different phenotypes in vivo are associated with ATPase motif mutations in Schizosaccharomyces pombe minichromosome maintenance proteins. Genetics 160: 1305–18. Johnson, R. E., R. Klassen, L. Prakash, S. P. Correspondence, and S. Prakash, 2015 A Major Role of DNA Polymerase δ in Replication of Both the Leading and Lagging DNA Strands. Mol. Cell 59: 163–175. Kunkel, T. A., and P. M. Burgers, 2008 Dividing the workload at a eukaryotic replication fork. Trends Cell Biol. 18: 521–7. Masai, H., C. Taniyama, K. Ogino, E. Matsui, N. Kakusho et al., 2006 Phosphorylation of MCM4 by Cdc7 kinase facilitates its interaction with Cdc45 on the chromatin. J. Biol. Chem. 281: 39249-61. Miyabe, I., T. a. Kunkel, and A. M. Carr, 2011 The major roles of DNA polymerases epsilon and delta at the eukaryotic replication fork are evolutionarily conserved. PLoS Genet. 7.(12) Miyabe, I., K. Mizuno, A. Keszthelyi, Y. Daigaku, M. Skouteri et al., 2015 Polymerase δ replicates both strands after homologous recombination-dependent fork restart. Nat. Struct. Mol. Biol. 22 (11): 932–38. Nick McElhinny, S. a., D. a. Gordenin, C. M. Stith, P. M. J. Burgers, and T. a. Kunkel, 2008 Division of Labor at the Eukaryotic Replication Fork. Mol. Cell 30: 137–144. Roberts, S. A., M. S. Lawrence, L. J. Klimczak, S. A. Grimm, D. Fargo et al., 2013 An APOBEC cytidine deaminase mutagenesis pattern is widespread in human cancers. Nat. Genet. 45: 970–6. Sabatinos, S. A., and S. L. Forsburg, 2010 Molecular Genetics of Schizosaccharomyces pombe. Methods Enzymol. 470: 759–95 Sheu, Y.-J., J. B. Kinney, A. Lengronne, P. Pasero, and B. Stillman, 2014 Domain within the helicase subunit Mcm4 integrates multiple kinase signals to control DNA replication initiation and fork progression. Proc. Natl. Acad. Sci. U. S. A. 111: E1899–908. 179 Sheu, Y.-J., and B. Stillman, 2006 Cdc7-Dbf4 phosphorylates MCM proteins via a docking site- mediated mechanism to promote S phase progression. Mol. Cell 24: 101–13. Sheu, Y.-J., and B. Stillman, 2010 The Dbf4-Cdc7 kinase promotes S phase by alleviating an inhibitory activity in Mcm4. Nature. 463: 113–117. Smith, H. C., R. P. Bennett, A. Kizilyer, W. M. McDougall, and K. M. Prohaska, 2012 Functions and regulation of the APOBEC family of proteins. Semin. Cell Dev. Biol. 23: 258–68. Stephens, D. J., and G. Banting, 2000 The use of yeast two-hybrid screens in studies of protein:protein interactions involved in trafficking. Traffic 1: 763–768.

Abstract (if available)

Abstract In this thesis, I’m using Schizosaccharomyces pombe (fission yeast) as a model system to perform an analysis of Mcm4 protein. In Chapter 1, I describe a collaborative project characterizing mcm4-M68 and mcm4-degron. We report that the mcm4 degron mutant is unable to complete DNA replication in S phase, but despite the under-replication, the cells are able to divide at restrictive temperature and release forming structures similar to micronuclei. This was a novel finding and the first report of a micronucleus structure in a yeast system. ❧ In Chapter 2, I characterize a truncation mutant of mcm4 that has a temperature sensitive phenotype which also is sensitivity to Methyl methanesulfonate (MMS) an alkylation damaging agent. This mutant lacks 106 amino acids from its C terminus. We see that this mutant functions differently than the canonical mcm4 temperature sensitive alleles (mcm4 M68 & mcm4ts-dg) for temperature sensitivity. The MMS sensitive phenotype has not been observed in other mcm4 mutants, which makes this mutant novel and shows that the C terminus might have a role in maintaining the replisome structure and help with responding to MMS treatment. ❧ Finally, in Chapter 3, I describe a collaborative project in which I performed the analysis in the fission yeast system, of a mutation predicted to disrupt a novel MCM filament and its importance in cell growth and survival.

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Creator Ranatunga, Nimna Samarawickrema (author)

Core Title Different alleles of fission yeast mcm4 uncover different roles in replication

School College of Letters, Arts and Sciences

Degree Doctor of Philosophy

Degree Program Molecular Biology

Publication Date 07/28/2016

Defense Date 02/03/2016

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

Tag checkpoint,DNA replication,fission yeast,fork protection complex,MCM complex,mcm4,OAI-PMH Harvest

Format application/pdf (imt)

Language English

Contributor Electronically uploaded by the author (provenance)

Advisor Forsburg, Susan (committee chair), Aparicio, Oscar (committee member), Reisler, Hanna (committee member)

Creator Email nims84@gmail.com,ranatung@usc.edu

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c40-283720

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