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University of Southern California Dissertations and Theses
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Mechanism study of SV40 large tumor antigen atpase and helicase functions in viral DNA replication
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Mechanism study of SV40 large tumor antigen atpase and helicase functions in viral DNA replication
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Content
MECHANISM STUDY OF SV40 LARGE
TUMOR ANTIGEN ATPASE AND
HELICASE FUNCTIONS IN VIRAL DNA
REPLICATION
by
Xian Jessica Yu
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 PHILOSOPY
(MOLECULAR BIOLOGY)
December 2012
Copyright 2012 Xian Jessica Yu
ii
Epigraph
“When one door closes another door opens; but we so often look so long and so
regretfully upon the closed door, that we do not see the ones which open for us.”
-Alexander Graham Bell
iii
Dedication
To my beloved grandparents, parents, husband and daughter
iv
Acknowledgments
I will be forever grateful to my advisor, Professor Xiaojiang Chen. He’s such a
great mentor for both science and life. No matter how difficult the research is, he is
always there to help and support. During these years of PhD study, my critical thinking
ability, analytical skill and problem-determination intelligence would not have grown
strong without him. The study progress of SV40 large tumor antigen also would not have
gone this far. Thank you XJ for guiding me on the tough science road.
I would also like to thank my awesome colleagues during the science discovery.
Thank you Dr. Ian Haworth and Dr. Susan Forsburg for being my thesis committee
members. Thank you Dr. Dahai Gai for teaching me all kinds of experimental techniques
and precious advices on research; Thank you Dr. Ganggang Wang and Dr. Etienne Toko
for teaching me helicase assay; Thank you Dr. Bo Zhou for being my best friend and
many encouragements when my science didn’t go well; Thank you Dr. Paul Chang and
Dr. Meng Xu for many wonderful discussions and research support; Thank you Dr.
Stanley Shi for fantastic statistical simulations; Thank you Ian Slaymaker for making
great structure figures. Thank you Yunxiang Mu for helping with DNA binding
Anisotropy. Thank you Dr. William B. Greenleaf and Joey Shi for great efforts in mutant
doping study. Thank you Dr. Sophia Tsai and Dr. Michelle Gomes for being awesome
carpool buddies. Thank you Dr. Michael Klein, Dr. Yi Zhang, Dr. Uday Sen, Dr. Jhimli
Dasgupta, Dr. Ronda Bransteitter, Dr. Courtney Prochnow, Dr. Aaron Brewster, Dr.
Lauren Holden, Shen Soh, Diana Wang, Yang Fu, Shi Jin, Lyon Chen, Xiao Xiao,
v
Maocai Yan, Damian Wang, Carolyn Truong, Samir Afif, Stephan Vila, Dorothy Wang
and Hasan Abbas for being my great lab friends during past and current years, as well as
your kindly help and support.
Importantly, I would like to thank my dear families and friends for their endless
love and support. Thank you Daddy and Mommy for providing me good education
otherwise I could not have made it this far. I feel sorry for leaving you two alone for
many years when I was studying aboard. I more and more realized that parents’ love is
the greatest love in the world since my first daughter came to this world two years ago.
Thank you my great husband Haiyue for supporting my study. You take family
responsibilities, take care of our daughter and make me free from housework. You are my
best soul mate and the one I want to spend the rest of life with. I also want to give my
daughter Chloe a special thank. You are such an amazing little angle that Mommy learns
love and patience from you. Thank you for being great when I was writing the thesis.
Thank you Tingting Lu, Liang Cheng, Fei Chen and Qingting He for being my forever
friends. I miss you all.
Lastly, I want to say sorry to my grandfathers for not being around when you both
left us. I finally made it! Thank you for always being proud of me. Wish you rest in
peace!
vi
Table of Contents
Epigraph ii
Dedication iii
Acknowledgments iv
List of Tables vii
List of Figures viii
Abstract ix
Chapter 1: Introduction 1
1.1 SV40 DNA Replication
1.2 Domains and Structure Overview of SV40 LTag
1.3 ATPase and Helicase Functions of SV40 LTag
1.4 Overview of Chapters
1
3
6
9
Chapter 2: Mechanism of Subunit Coordination of A Hexameric
Molecular Machine
11
2.1 Introduction
2.2 Results
2.3 Discussion
2.4 Experimental Procedures
12
14
27
34
Chapter 3: Mechanisms of β-hairpin and F-loop Structures of Simian
Virus 40 Hexameric Helicase in DNA Replication Initiation
and DNA Unwinding
44
3.1 Introduction
3.2 Results
3.3 Discussion
3.4 Experimental Procedures
45
50
64
73
Bibliography 79
vii
List of Tables
Table 2.1: Biochemical properties of WT and mutant LTag 17
Table 2.2: DNA substrates used in doping ATPase and helicase Assays 40
Table 2.3: Deduction of activity in different models 41
Table 3.1: Residues used to substitute the β-hairpin and F-loop residues 48
Table 3.2: DNA substrates used in assays 75
viii
List of Figures
Figure 1.1: SV40 DNA genome, core origin sequence and eukaryotic
DNA replication model 4
Figure 1.2: LTag functional domains and domain structures 6
Figure 1.3: Structural view of ATP-pockets and β-hairpin on LTag
AAA+ helicase domain 8
Figure 2.1: ATP-pockets and central channel β-hairpin on LTag AAA+
helicase domain 16
Figure 2.2: Biochemical characterizations of the tri-cis mutant and β-
hairpin mutant 19
Figure 2.3: ATPase doping assay of LTag with tri-cis mutant in the
absence or presence of different DNAs 23
Figure 2.4: Helicase doping assay with LTag tri-cis mutant 25
Figure 2.5: Helicase doping study with LTag β-hairpin mutant
containing K512A/H513A mutations 27
Figure 3.1: SV40 LTag domain structure and the various activities 49
Figure 3.2: DNA binding anisotropy of LTag WT and mutants 52
Figure 3.3: ATPase stimulation assay in the presence of ssDNA or
dsDNA 55
Figure 3.4: Unwinding activity of LTag mutants on fork-shaped DNA 57
Figure 3.5: Unwinding activity of LTag mutants on origin dsDNA 58
Figure 3.6: Helicase-mediated biotin-streptavidin displacement assay on
ssDNA 60
Figure 3.7: Helicase-mediated biotin-streptavidin displacement assay on
dsDNA 61
Figure 3.8: Potassium permanganate reactivity assay 63
ix
Figure 3.9: Activity summary of mutations on β-hairpin and F-loop
structures 65
Figure 3.10: Interactions of residues on β-hairpin and loop structures
with EP-origin DNA 71
x
Abstract
Simian Virus 40 (SV40) has been studied as the model system to elucidate the
mechanism of eukaryotic DNA replication. The oncogenic large tumor antigen (LTag)
encoded by SV40 not only transforms cells and induces tumors but also functions as a
molecular motor machine that melts the viral origin and unwinds duplex DNA to initiate
replication. It’s been regarded as the functional homologue of minichromosome
maintenance (MCM) protein, a putative replicative helicase in eukaryotic and archaeal
cells.
Simian virus 40 large tumor antigen (LTag) is an AAA+ hexameric motor that
harnesses the energy from ATP binding/hydrolysis to initiate DNA replication and
unwind replication forks. However, how the six subunits of LTag hexamer motor
coordinate for ATP hydrolysis and for DNA unwinding/translocation are unresolved.
Here I investigated the subunit coordination mechanisms for ATP hydrolysis and DNA
unwinding through a series of mutant doping experiments. For ATP hydrolysis, I
observed a random mode in the absence of DNA, a semi-random mode with ssDNA, and
a semi-coordinated mode with fork or origin DNA. For DNA unwinding, however, the
results indicated a semi-coordinated mode for fork-DNA, but a fully coordinated mode
for origin DNA. These results and previous evidence suggest a distinctive coordination
behavior for LTag, which adopts different coordination for ssDNA translocation, fork-
DNA unwinding, and origin DNA unwinding. For origin DNA unwinding, LTag
hexamer operates in a fully coordinated mode.
xi
The β-hairpin and F-loop in the central channel of LTag hexamer has been
identified to play a key role in interacting with DNA substrate. Here, I made a series of
mutations on the tip of β-hairpin and an adjacent F-loop structure. A variety of functional
assays have been performed using these mutants. I demonstrate that two neighboring β-
hairpin tip residues (K512 and H513) and residue F459 on F-loop are involved in DNA-
dependent ATPase stimulation, in local origin melting, ss- and ds- DNA translocation,
and fork- and origin- DNA unwinding. However, their relative functional roles in these
activities are different, with K512 as a sensor to recognize and convey the DNA signal
from the central channel while H513 and F459 as mechanics to interact with DNA base
pairs for melting and unwinding. The biochemical results support the structural
observations that the positively charged residue K512 interacts with DNA phosphate
backbone through electrostatic interactions while ring-shaped residues H513 and F459
interact with DNA by hydrophobic stacking.
In summary, my thesis is focused on the functional study of LTag in regard to
ATP hydrolysis and DNA unwinding, as well as the role defining of important residues
on β-hairpin and loop structure. The overall work will provide important clues towards
studying other motor helicases in the same family and insights into the big picture of
DNA initiation and replication.
1
Chapter 1
Introduction
1.1 SV40 DNA Replication
Simian Virus 40 (SV40), a member of the Polyomavirus family, was first
identified as a contaminating virus in rhesus macaque monkey cells, which were used to
produce polio vaccine (Hilleman, 1998). Later, it was determined that SV40 virus causes
a variety of tumors in hamsters and rats (Eibl et al., 1994). However, it’s still in debate
whether SV40 has an etiologic role of human cancer even though the presence of SV40
DNA and gene expression have been proven in some human tumors.
During the development of modern molecular biology, SV40 has been used as a
model system to study a variety of molecular processes including genome replication,
gene expression, posttranscriptional process and cell cycle regulation (Sullivan and Pipas,
2002). SV40 DNA replication is the best understood eukaryotic replication system to date.
SV40 DNA replication takes place in the nucleus of the host cell and the SV40 genome is
organized into minichromosome containing host cell histones. With the exception of
SV40 encoded protein large T antigen (LTag), all the proteins required for SV40 DNA
replication are supplied from the host cell (Kelly, 1988). Furthermore, SV40 genome has
one specific origin of replication and the origin sequence has been well characterized
2
(Danna and Nathans, 1972; Dean et al., 1987). Therefore, SV40 serves as a simple and
powerful tool to understand complicated eukaryotic cell growth and development.
With the help of restriction site and physical map, a 5243bp sequence of SV40
DNA was determined in 1978 as the first completely sequenced eukaryotic genome
(Danna and Nathans, 1972; Fiers et al., 1978; Nathans and Danna, 1972; Reddy et al.,
1978). SV40 has a simple architecture of genome, which contains seven gene products
plus a single replication origin within a covalently closed double-stranded DNA (Fig.
1.1A). The early gene products - large T antigen (LTag), small t antigen, and 17kT
antigen – are encoded by three alternatively spliced products and share 82 amino acids at
the amino-terminal (Brodsky and Pipas, 1998). These regulatory proteins are involved in
early and late gene transcription, induction of host cell transcription, viral DNA
replication and virion assembly. Agno protein is expressed late in infection and may
function in assembly and/or release of viral capsid. Three late genes products – VP1, VP2
and VP3 - are the structure proteins that form viral capsid (Crawford and Black, 1964;
Dulbecco and Vogt, 1963). Further study of the viral replication origin revealed a 64bp
core origin, which is required to initiate viral DNA replication. The origin contains three
segments: a central element containing four pentanucleotides (GAGGC), an early
palindrome (EP) sequence and an AT- rich tract (Fig. 1.1B).
The only SV40-encoded replication protein LTag recognizes the origin and forms
a double-hexamer on it (Joo et al., 1998). The binding sites have been identified within
four pentanucleotides. The binding of double hexamer on origin induces the structure
3
bending and distortion within origin DNA: the melting of 8bp at the EP region and the
unwinding/distortion of the AT-rich sequence (Borowiec and Hurwitz, 1988). To
continue the initiation process, LTag recruits cellular proteins: replication protein A
(RPA), DNA polymerase α/primase and topoisomerase I (topo I) (Collins and Kelly,
1991; Murakami et al., 1986; Trowbridge et al., 1999). LTag functions as a helicase
during the DNA replication by separating duplex DNA and the unwinding is powered by
ATP hydrolysis. The unwinding reaction established two replication forks and generates
the substrate for cellular proteins to continue the elongation stage of the replication. DNA
polymerase δ, proliferating cell nuclear antigen (PCNA), replication factor C (RF-C) and
other cellular replication proteins assemble to the replication fork orderly for DNA
synthesis (Bylund et al., 2006; Garg and Burgers, 2005; Oku et al., 1998; Pavlov et al.,
2006) (Fig. 1.1C). By addition of purified replication proteins and LTag to SV40 origin
DNA, the SV40 replication process can be reconstituted in vitro in a cell-free system,
which facilitates the study of proteins involved in eukaryotic replication and regulation
(Hurwitz et al., 1990; Li and Kelly, 1984).
1.2 Domains and Structure Overview of SV40 LTag
SV40 LTag is the only viral-encoded protein participated in viral DNA replication
in the host cell. LTag is a multifunctional protein that transforms cells, initiates
replication, unwinds duplex DNA and recruits other cellular replication proteins
(Borowiec et al., 1990; Dean et al., 1987; Stillman et al., 1985). It’s a functional
4
homologue of eukaryotic minichromosome maintenance (MCM) protein, a putative
replicative helicase in eukaryotic and archaeal cells (Chong et al., 2000; Fletcher et al.,
2003; Kelman et al., 1999).
Figure 1.1 SV40 DNA genome, core origin sequence and eukaryotic DNA replication
model. (A) SV40 genomic organization of the alternatively spliced early (large T antigen
[LT], small t antigen [St], and 17k T antigen) and late (VP1 to 3) proteins. The early (PE)
and late (PL) promoters exist in opposite orientations that flank the SV40 origin (Ori) of
replication (Sullivan and Pipas, 2002). (B) The SV40 64-bp core origin sequence. The
pentanucleotides P1 through P4 are indicated above the sequence. Each GAGGC
sequence is colored magenta, and its complement is cyan. The arrows indicate the 5’-3’
direction of the pentanucleotide sequence GAGGC. The AT-rich and early palindrome
regions of the SV40 core origin are labeled. (C) A minimal set of replication proteins at a
eukaryotic fork. MCM helicase substitutes here for LTag; the PCNA clamp loader RFC
and topoisomerases are not shown (Fanning and Zhao, 2009).
5
LTag has 708 residues and folds into three major domains: DnaJ domain (residue
1-82), origin binding domain (OBD) (residue131-259) and helicase domain (251-627)
(Arthur et al., 1988; Campbell et al., 1997; Li et al., 2003) (Fig. 1.2A). The N-terminal J
domain shares the sequence homolog with the J domain of the DnaJ (Hsp40) family of
molecular chaperones. As one of the transforming domains of LTag, J domain binds and
stimulates the ATPase activity of specific Hsp70/DnaK family members. J domain is
dispensable for in vitro DNA replication (DeCaprio, 1999; Sullivan and Pipas, 2002).
The crystal structure of N-terminal region of LTag (residue 7-117) in complex with
retinoblastoma (Rb) pocket domain provided insights into how LTag inactivates tumor
suppressor during tumorigenesis (Kim et al., 2001). The OBD recognizes the origin and
is required for LTag assembly at origin (Arthur et al., 1988). The structure study of OBD
alone or with origin DNA has revealed the mechanism of origin recognition by LTag and
its assembly of double hexamer (Bullock, 1997; Joo et al., 1997). The helicase domain is
comprised of two subdomains: Zn-binding domain and ATPase domain. The first crystal
structure of LTag helicase domain was determined by Xiaojiang Chen’s laboratory and
provided the mechanism of LTag on DNA unwinding and translocation (Li et al., 2003).
Zn-binding domain is important in LTag hexamerization. The ATPase domain involves
in helicase function of LTag by interacting with DNA through the central channel. LTag
helicase domain also serves a docking site for binding of tumor suppressor p53 and other
cellular replication proteins (Lilyestrom et al., 2006). Remarkably, the overall structure of
LTag helicase domain is closely related to the architecture of other replicative helicases,
e.g. archeal MCM (Brewster et al., 2008; Fletcher et al., 2003) (Fig.1.2B).
6
Figure 1.2 LTag functional domains and domain structures. (A) The schematic
representation of LTag functional domains. The binding proteins are listed n the top of
each domain (Gai et al., 2004a). (B) Modular organization of Tag domains and linkers.
Atomic structures of DnaJ (Kim et al., 2001), OBD (Luo et al., 1996), and helicase (Zn
and ATPase/AAA+) domains (Li et al., 2003) are shown approximately to scale with the
intervening peptides as dotted lines. The structure of the host-range domain has not been
determined (Fanning and Zhao, 2009).
1.3 ATPase and Helicase Functions of SV40 LTag
To study LTag function as a ATPase and a helicase, two structures on LTag
helicase domain are the focus: ATP-binding pocket and central channel β-hairpin/loop
(Fig. 1.3A). The active form of LTag is a ring hexamer triggered by ATP binding. There
are six ATP-binding pockets between two adjacent monomers. The residues on both
7
monomers (cis- and trans-) interact with nucleotide (Nt) base, phosphate and pentose
moiety (Fig. 1.3B). As a typical AAA+ family member, LTag couples ATP
binding/hydrolysis to its DNA remolding work. How does nucleotide hydrolysis couples
to unwinding and translocation in hexameric helicases has been a hot study subject over
many years. Based on an all-or-none Nt binding mode in LTag-Nt co-crystal structure, a
concerted ATP hydrolysis mode has been proposed, which indicates that all six
monomers bind, hydrolyze and release ATP simultaneously (Gai et al., 2004b). However,
the biochemical data to support this conclusion is still needed and the work in this
dissertation provides such data.
The ATP binding, hydrolysis and release causes conformational changes of the
hexamer. The most obvious changes are the central channel dimension shifting from 14Å
to 22Å upon the ATP binding to ADP binding and to Nt-free state. Within the central
channel, there are six positively charged β-hairpins and F-loops, which directly interact
with DNA (Fig. 1.3C). The different ATP binding states also made a longitudinal
movement of β-hairpins along the central channel about 17Å (Gai et al., 2004b). Based
on the observations, a looping model for unwinding has been proposed that showing the
coupling of the β-hairpin movement to the dsDNA translocation into LTag hexamer for
unwinding. It also addressed the importance of β-hairpin residues in interacting with
DNA during unwinding. Recent new co-crystal structure captured an origin melting state
of LTag with origin dsDNA in the central channel (Chang et al., 2012). The detailed
interacting residues were identified as β-hairpin tip residues K512, H513 and F-loop
residues. Although the previous biochemical study has done to discover the roles of β-
8
hairpin and loop residues in helicase function (Borowiec et al., 1990; Shen et al., 2005),
the other functions such as ATPase stimulation, origin melting and translocation are still
elusive due to no solid biochemical data. In this book, I describe a detailed mutagenesis
study of β-hairpin and loop residues in a variety of activities that LTag involves in as a
replication initiator.
Figure 1.3 Structural view of ATP-pockets and β-hairpin on LTag AAA+ helicase
domain. (A) The LTag hexamer structure viewing from C-terminal end, showing the six
ATPs (in yellow) bound between two adjacent subunits (in silver and black), and the β-
hairpin tip residues K512 and H513 (in red) in the central channel. (B) A close-up view
of an ATP-pocket between two neighboring subunits (in silver and black). Three cis-
residues (in blue) on the cis subunit (in black) that has the p-loop and three trans-residues
(in cyan) on the trans subunit (in silver) for interacting with the ATP are shown. (C) A
close-up view of the K512 and H513 residues on the central channel β-hairpin tips (in
red).
9
1.4 Overview of Chapters
The structure study of LTag domains provides insights into LTag hexamerization,
DNA binding, origin melting, ATP hydrolysis, and helicase functions. The biochemical
study verifies the observations in the structure and provides the understanding of how the
molecular motor machine works. This thesis majorly focuses on the biochemical
characterization of LTag as an ATPase and helicase during DNA replication.
In chapter 2, I investigated the subunit coordination mechanisms for ATP
hydrolysis and DNA unwinding through a series of mutant doping experiments. For ATP
hydrolysis, I observed a random mode in the absence of DNA, a semi-random mode with
ssDNA, and a semi-coordinated mode with fork or origin dsDNA. For DNA unwinding,
the results indicated a semi-coordinated mode for fork-DNA, but a fully coordinated
mode for origin dsDNA. These results and previous structural evidence suggest that LTag
adopts different subunit coordination for ssDNA translocation, fork-DNA unwinding, and
origin DNA unwinding. For origin DNA unwinding, LTag hexamer operates in a fully
coordinated mode, likely in a concerted manner.
In chapter 3, I made a series of mutants of the residues on the tip of β-hairpin and
an adjacent F-loop structure. A variety of functional assays have been performed using
these mutants, including ATPase stimulation, local origin melting, and DNA unwinding
and translocation. I demonstrate that two neighboring β-hairpin tip residues K512 and
H513 are involved in DNA-dependent ATPase stimulation, in local origin melting, ss-
and ds- DNA translocation, and fork- and origin- DNA unwinding. However, their
10
relative functional roles in these activities are different, with K512 being the most
stringent, in which any substitution of the Lys residue kills the function. On the other
hand, H513 and F459 can be substituted by ring shaped residues and still fulfill the
function. I have also identified mutants that either uncouple the ATPase activity from
DNA translocation and unwinding, or uncouple the origin melting from DNA unwinding.
These biochemical studies offer new opportunities to understand the molecular
details of the origin DNA melting, ATP hydrolysis and unwinding, an essential process
for the initiation of DNA replication.
11
Chapter 2
Mechanism of Subunit Coordination of A
Hexameric Molecular Machine
Reproduced with permission from Xian Jessica Yu, William B. Greenleaf, Yemin Stanley
Shi, Dahai Gai, Xiaojiang S. Chen 2012 Mechanism of Subunit Coordination of A
Hexameric Molecular Machine (Manuscript submitted).
Author contributions: X.J.Y. purified proteins and DNA substrates, carried out all the
biochemical doping experiments and analyzed the data; W.B.G. made tri-cis mutant
construct and carried out doping ATPase assay without DNA and with ssDNA; Y.S.S.
designed the statistical simulation model; D.G. provided advice for project design and
data analysis and X.S.C. supervised the project.
Simian virus 40 large tumor antigen (LTag) is an AAA+ hexameric motor that harnesses
the energy from ATP binding/hydrolysis to initiate DNA replication and unwind
replication forks. However, how the six subunits of LTag hexamer motor coordinate for
ATP hydrolysis and for DNA unwinding/translocation are unresolved. Here I
investigated the subunit coordination mechanisms for ATP hydrolysis and DNA
unwinding through a series of mutant doping experiments. For ATP hydrolysis, I
observed a random mode in the absence of DNA, a semi-random mode with ssDNA, and
12
a semi-coordinated mode with fork or origin DNA. For DNA unwinding, however, the
results indicated a semi-coordinated mode for fork-DNA, but a fully coordinated mode
for origin DNA. These results and previous evidence suggest a distinctive coordination
behavior for LTag, which adopts different coordination for ssDNA translocation, fork-
DNA unwinding, and origin DNA unwinding. For origin DNA unwinding, LTag
hexamer operates in a fully coordinated mode.
2.1 Introduction
Simian virus 40 (SV40) Large Tumor antigen (LTag) is an AAA+ hexameric
motor protein that functions as the helicase essential for viral DNA replication in
mammalian cells (Borowiec et al., 1990; Stillman and Gluzman, 1985; Sullivan and
Pipas, 2002). It recognizes the specific origin DNA sequence, assembles as a double
hexamer and melts the origin DNA, unwinds the replication forks, and recruits DNA
polymerase/primase and other cellular replication proteins (Dean et al., 1987; Stillman,
1994). These multiple functions allow LTag to serve as a good and simple model system
to study eukaryotic DNA replication, and LTag is considered a functional homologue of
the minichromosome maintenance (MCM) protein in eukaryotic and archaeal cells
(Chong et al., 2000; Fletcher et al., 2003; Kelman et al., 1999).
As a typical AAA+ family member, LTag in a hexamer form can couple the
energy of ATP binding/hydrolysis to the mechanical work for unwinding and/or
translocation of nucleic acids. The six subunits in a hexameric ring reconstitute six active
13
sites for ATP hydrolysis. How the six subunits of LTag coordinate with each other in the
ring-shaped hexamer for ATP hydrolysis is unknown, even though previous crystal
structures in different nucleotide binding states showed that all six subunits of LTag has a
all-or-none nucleotide binding mode, suggesting a highly concerted mode of action (Gai
et al., 2004b).
However, this proposed concerted mode is at odds with the various cooperative
models for four other hexameric motors characterized so far, Sulfolobus solfataricus
MCM (SsoMCM), T7 gp4, and BPV E1, and ClpX. The biochemical data for SsoMCM
indicates no subunit coordination for ATP hydrolysis and limited coordination for DNA
unwinding (Moreau et al., 2007). T7 gp4 shows a fully coordinated dTTP hydrolysis in
the presence of ssDNA, which is consistent with a strict sequential propagation for dTTP
hydrolysis based on biochemical data and on structures showing partial occupancy of
nucleotide pockets (Bird et al., 2000; Crampton et al., 2006; Donmez and Patel, 2006;
Singleton et al., 2000). The structure of ssDNA bound BPV E1 helicase showed ssDNA
contacts all subunit β-hairpins in a helicase manner, in an apparent agreement with the
sequential mode for T7 gp4 (Enemark and Joshua-Tor, 2006). Previous evidence
indicates ClpX, a protein unfolding hexameric motor, uses a random (or probabilistic)
mode for ATP hydrolysis (Martin et al., 2005).
For LTag, the all-or-none nucleotide binding mode observed in LTag structures
could be the results of particular conformations favored for crystal packing, and thus may
not necessarily be a manifestation of the real subunit coordination mode for ATP binding
14
and hydrolysis. In order to understand how the six subunits of LTag coordinate for ATP
hydrolysis to drive the unwinding function, I performed biochemical studies to examine
the subunit coordination mechanisms under various substrate conditions.
In the present study, I investigated the subunit coordination around the LTag
hexamer ring for both the ATP hydrolysis and for the helicase function using the mutant
doping method. Our data reveals that the subunit coordination modes for both ATP
hydrolysis and DNA unwinding not only depend on the presence of DNA, but also
depend on the types of DNA substrates. For ATP hydrolysis, it shows a random mode in
the absence of DNA, a semi-random mode in the presence of ssDNA, and a semi-
coordinated mode in the presence of fork or origin DNA. For DNA unwinding, our
results shows fully coordinated mode when assayed with the origin DNA, which suggests
a different working mechanism for the unwinding of fork DNA and origin DNA.
2.2 Results
Generation of Mutants Suitable for Doping Experiment
In order to understand the coordination mechanisms between subunits within the
ring-shaped hexamers of LTag, I used the so-called mutant “doping” method, by which
increasing amounts of non-catalytic mutant are titrated into WT protein, and the ATPase
and helicase activities of the resulting hexamers containing increasing number of mutant
are measured. The key is to generate non-catalytic mutants with no residual ATPase
15
activity and helicase activity in order to have a clean background, but still with normal
level of hexamerization activity with itself and with WT protein.
To completely abolish ATPase activity, I made two mutants that were designed
based on the high-resolution structure of LTag (Gai et al., 2004b). One is the tri-cis
mutant (cT434A/cD474A/cN529A), in which three “cis” residues around the ATPase
pocket were mutated (Fig. 2.1B). These three cis residues interact with the triphosphate
of ATP through Mg
++
and water-mediated hydrogen bonds (Fig. 2.1B). Another is tri-
trans mutant (tK418A/tR498A/tR540A), in which Lys/Arg finger residues on a “trans”
subunit that interact with the ATP-pocket of an adjacent subunit (cis subunit) are
mutated.
The ATPase assay of both mutants showed no detectable ATPase activity (Table
2.1), indicating that both mutants completely knock out ATP hydrolysis, satisfying one of
the conditions for doping experiment. I next examined if the mutations affected
hexamerization using gel filtration chromatography. LTag equilibrates between monomer
and hexamer in solution (Gai et al., 2004a). ATP promotes hexamer formation due to
ATP binding at the interface between a cis and a trans subunits, and thus presence of
ATP will increase hexamer-to-monomer peak ratio in gel filtration chromatography. The
results showed that the tri-trans mutant oligomerized poorly compared to wild-type LTag
(Table 2.1), indicating that this mutant is not suitable for mutant doping experiment.
16
Figure 2.1 ATP-pockets and central channel β-hairpin on LTag AAA+ helicase
domain. (A) The LTag hexamer structure viewing from C-terminal end, showing the six
ATPs (in yellow) bound between two adjacent subunits (in silver and black), and the β-
hairpin tip residues K512 and H513 (in red) in the central channel. (B) A close-up view
of an ATP-pocket between two neighboring subunits (in silver and black). Three cis-
residues (in blue) on the cis subunit (in black) that has the p-loop and three trans-residues
(in cyan) on the trans subunit (in silver) for interacting with the ATP are shown. (C) A
close-up view of the K512 and H513 residues on the central channel β-hairpin tips (in
red). (D) The structural connection between the β-hairpin in the central channel and the
ATP-pocket, as well as between two ATP sites in a hexamer, providing a structural basis
for functional coupling between the two structural elements.
17
Table 2.1. Biochemical properties of WT and mutant LTag
Hexamer:Monomer
peak ratio
a
Catalytic
activity
ssDNA
binding
Enzyme −ATP +ATP k
cat
, min
-1
K
m
, µM K
d, app
, nM
WT
0.3
0.5
20 ± 1
270 ± 40
180 ± 10
Triple-trans 0.04 0.06 −
b
−
b
nd
Triple-cis 0.2 0.5 −
b
−
b
210 ± 10
a
Hexamer:Monomer peak ratios were calculated based on peak areas after analyzing 9 nmol of protein on
S200 analytical column (GE) in the presence and absence of ATP. ATP is known to promote hexamer
formation, and increase the ratio of hexamer:monomer for wild type. The result shows that Triple-cis
mutant has the hexamerization and DNA binding activities comparable to those of the WT;
b
only
background activity; nd: not determined
However, the tri-cis mutant has the WT-level hexamerization activity in the
absence or presence of ATP (Table 2.1). Thus the tri-cis mutant was further examined for
its ability to associate with WT subunits using GST pull-down assay. Purified WT GST-
LTag was incubated with non-tagged mutant or WT LTag in the presence of ATP that is
known to promote stable hexamer formation. The mixture was then passed through a
glutathione sepharose resin. After extensive washing with binding buffer, the resin was
analyzed by SDS-PAGE to detect the non-tagged protein retained by the GST-tagged
protein on the resin. The results showed that the WT GST-LTag had the same ability to
pull down non-tagged mutant and WT (Fig. 2.2A, lanes 2-6). Conversely, using the
mutant GST-LTag to pull down non-tagged mutant and WT gave the same results (Fig.
18
2.2B). These data suggest that tri-cis mutant interacts with WT protein just as well as the
WT interacts with itself, therefore it satisfies the criteria as a true non-catalytic subunit
that retained WT-level of hexamerization activity, making it suitable for our mutant
doping experiment to study subunit coordination for ATP hydrolysis and DNA
unwinding.
In addition to the ATPase-defective tri-cis mutant for studying the subunit
coordination, I also generated a β-hairpin mutant with mutations on the β-hairpin tip
residues, K512A/H513A. These two residues are important for DNA interactions in the
central channel and for DNA unwinding (Kumar et al., 2007; Li et al., 2003; Shen et al.,
2005). These β-hairpin residues are distal to the ATP-binding pocket, and thus should
not disrupt the intrinsic ATP hydrolysis ability of LTag. Indeed, our assay results showed
that, unlike the tri-cis mutant that completely lost the ATPase activity, the β-hairpin
mutant had ATPase activity comparable to the WT in the absence of DNA (Fig. 2.2C).
However, this mutant failed to show elevated ATPase activity in the presence of DNA
(Fig. 2.2C), which likely is because this mutant affected DNA binding in the central
channel (Kumar et al., 2007; Shen et al., 2005), thus affecting the DNA-stimulated
ATPase function. The hexamerization assay showed this mutant hexamerized as well as
WT, and GST pull down assay demonstrated that this mutant binds to WT subunits just
as well as WT bind to itself (Fig. 2.2A, lanes 2-4, 7-8; Fig. 2.2B). This β-hairpin mutant
is used to evaluate the subunit coordination for DNA unwinding in the helicase doping
assay.
19
Figure 2.2 Biochemical Characterizations of the tri-cis Mutant and β-hairpin
Mutant. (A) GST-WT LTag pull down assay, showing that GST-WT pulled down non-
tagged WT, tri-cis and β-hairpin mutants equally well. (B) GST-mutant LTag pull down
assay, showing GST-tri-cis and GST-β-hairpin pulled down non-tagged self and WT
equally well. The results in (A) and (B) indicate the mutants retain the ability to associate
with each other and with WT to hexamerize. (C) ATPase activity assay in the
absence/presence of DNAs. The tri-cis mutant showed a complete loss of activity, and the
β-hairpin retained WT-level activity in the absence of DNA, but lost DNA-stimulated
activity. (D, E) Helicase assay, showing the tri-cis and β-hairpin mutants are helicase
defective for fork-DNA substrate (D) and blunt-ended origin DNA (origin DNA) (E). B:
Boiled DNA; UB: unboiled DNA with no protein. The protein in lanes 1, 3, 5 was 0.11
µM and in lanes 2, 4, 6 was 0.22 µM as monomers.
20
Subunit Coordination for ATP Hydrolysis: Doping with ATPase Mutant
Having obtained the non-catalytic tri-cis mutant, I performed the mutant doping
experiment to characterize the coordination mode of the six subunits for ATP hydrolysis
in the absence and presence of various types of DNA. In the doping assay, how the six
subunits in a hexamer coordinate with each other for a specific activity, such as ATP
hydrolysis, can be analyzed based on the ATPase activity changes when the WT protein
is titrated with increasing amount of non-catalytic mutant.
Two extreme cases for the coordination mode on ATP hydrolysis are as follows:
first is the random mode, in which no coordination exists between the six sites, and each
site hydrolyzes ATP independent of the other sites in the hexamer. In the random mode, a
hexamer can hydrolyze ATP even if it contains only one WT subunit, and the ATPase
activity decreases proportionally to the percentage of WT subunits. The second extreme
case is the full coordination mode, in which ATP hydrolysis at one site is dependent on
all the other sites in the hexamer, and ATP hydrolysis can only occur when all six
subunits in the hexamer are WT. In the full coordination mode, ATPase activity
decreases exponentially as the percentage of WT subunits decreases. Between these two
extreme cases, there are other possible intermediate coordination modes, some of which
can be described by the mathematical model listed in Eq. 1 (also see Experimental
Procedures and Table 2.3). The possible cases predicted by Eq. 1 are listed in Table 2.3.
21
Y= (1)
Using the doping method, in which an increasing percentage of the non-catalytic mutant
is titrated into the WT, I measured activity changes in response to the increasing
percentage of mutant and plotted these against the modeled curves predicted by Eq. 1.
The modeled curves for all the six possibilities predicted by Eq. 1 are drawn in different
colors in Fig. 2.3, 2.4 and 2.5, which are shown as C=1, 2…6. In these figures, the
experimentally measured activity data are shown as dots on top of these curves, which
will be discussed in details below.
ATPase activity of LTag containing different ratios of WT and mutant was
measured in the absence of DNA or presence of different types of DNA (ssDNA, fork
DNA, and blunt-ended origin containing DNA) (Fig. 2.3A-D). In the absence of DNA, as
the percentage of tri-cis mutant increased, the ATPase activity decreased proportionately
to the percentage of WT, which fits well with the modeled curve of linear decrease (C=1)
(Fig. 2.3A). This result indicates that, in the absence of DNA, the six subunits in a LTag
hexamer hydrolyze ATP independent of each other, or in a random mode.
Then I tested the LTag ATPase activity in the presence of ssDNA. LTag can bind
and translocate on ssDNA (Morris et al., 2002). Compared with the reaction without
DNA, the ATPase activity here decreased more rapidly as the WT was titrated out by the
tri-cis mutant in the presence of ssDNA (Fig. 2.3B), moving away from the C=1 linear
curve (red line) and towards the C=2 (green) curve (Fig. 2.3B). This indicates the six
22
subunits in the LTag hexamer no longer hydrolyze ATP randomly. To distinguish from
the linear C=1 random mode, I call this a semi-random mode. This result is distinctive
from T7 gp4 that showed a random mode for nucleotide hydrolysis in the absence of
DNA (a linear decrease of activity) was switched to a fully coordinated mode when
ssDNA was present (an exponential decrease of activity) (Crampton et al., 2006).
Next I examined the ATP hydrolysis in the presence of two types of dsDNA, a
fork DNA and a blunt-ended dsDNA containing SV40 origin sequence (origin DNA),
both of which can be unwound by LTag. I observed clearly different coordination modes
in ATP hydrolysis with these dsDNA when compared with ssDNA. As can be seen in
Figs. 2.3C and 2.3D, the decrease in ATPase activity followed the C=3 model (blue
curves) for both types of dsDNA, a much more rapid decrease than when ssDNA is used
(Fig. 2.3B). These results indicate that the presence of dsDNA somehow causes the
hexamer to hydrolyze ATP in a more coordinated manner. The C=3 model is called semi-
coordinated mode (to distinguish from a full coordinated mode). Taking the results
together from above, I conclude that DNA can switch the coordination mode for ATP
hydrolysis from a random mode to semi-random and to semi-coordinated modes, and that
different types of DNA can trigger different subunit coordination modes.
23
Figure. 2.3 ATPase doping assay of LTag with tri-cis mutant in the absence or
presence of different DNAs. For all panels, modeling of all possible coordination modes
was described in Experimental Procedures and in Table 2.3, and the modeled curves for
each possible mode (C=1, 2…6) are drawn in colored curves in panels A-D. (A) In the
absence of DNA substrate, ATPase doping result fits the C=1 linear simulated model (R
2
= 0.983) that is the random mode. (B) In the presence of ssDNA, the result shows a fit
between the C=1 linear and the C=2 “pairs” simulated model (green curve). This mode is
termed semi-random here. (C) In the presence of fork-DNA, the result fits the C=3
“Trimer” simulation or a semi-coordinated model (blue curve)(R
2
= 0.991), which
requires at least three active subunit for ATP hydrolysis. (D) In the presence of a blunt-
ended origin DNA, the result again fits a “Trimer” simulation (R
2
= 0.998). The error bars
in all panels represent the standard deviation of three independent experiments.
24
Subunit Coordination for DNA Unwinding: Doping with ATPase Mutant
Using the same doping approach, I observed helicase activity instead of ATPase
activity to study the subunit coordination for helicase function in the presence of fork
DNA and blunt-ended origin DNA as unwinding substrates. When using the fork DNA,
the helicase activity decreased rapidly as the tri-cis mutant titrate out the WT, which fits
well into the modeled curve for semi-coordinated mode (C=3, blue curve) (Fig. 2.4A).
This semi-coordinated mode for fork DNA unwinding is the same as the mode obtained
for ATP hydrolysis in the presence of fork DNA, but different from the semi-random
mode for ATP hydrolysis with ssDNA. This suggests that during fork DNA unwinding,
the energy produced by ATP hydrolysis is efficiently coupled to the helicase function to
result in a matching semi-coordinated mode. This semi-coordination mode requires at
least three active ATPase subunits in the ring hexamer to function (Table 2.3). This result
is different to the case for SSoMCM (Moreau et al., 2007), which displayed a less
coordinated mode termed as pairs-model (similar to the C=2 model here).
Surprisingly, when blunt-ended origin DNA was used as unwinding substrate, a
distinct coordination mode was revealed by the doping assay. The result showed that the
change of origin unwinding activity fits the exponential decrease (C=6, yellow curve in
Fig. 2.4B) when titrating the WT with the tri-cis mutant. The C=6 curve is a full
coordination model as predicted by Eq. 1, i.e. all six subunits in a hexamer need to be
WT to have activity, and a single non-catalytic subunit within a hexamer will kill the
activity completely. This result indicates that origin DNA unwinding requires full
25
coordination between six subunits, distinct from the semi-coordination mode for
unwinding fork DNA (compare Fig. 2.4A, 2.4B). This is the first direct biochemical
evidence showing a clear difference in unwinding fork DNA vs. origin DNA by a
hexameric helicase like LTag.
Figure 2.4 Helicase doping assay with LTag tri-cis mutant. As in Figure 2.3, the
modeled curves for each possible coordination mode (C=1, 2…6) for helicase activity are
drawn in colored curves in panels A-B. (A) In the presence of fork-DNA, helicase doping
result has the best fit for the C=3 “Trimer” simulation or a semi-coordinated model (blue
curve) (R
2
= 0.993), suggesting that unwinding the fork DNA requires at least three
active subunit in a hexamer. (B) In the presence of a blunt-ended origin-DNA, helicase
doping result fits an “Exponential” simulation curve (in yellow) (R
2
=0.993), which is a
full coordination model that requires all six subunits to be active to unwind the origin
DNA. Either a fully sequential or a fully concerted mode for the six subunits yields the
exponential simulation curve. The error bars represent the standard deviation of five
independent experiments.
26
Subunit Coordination for DNA Unwinding: Doping with β-Hairpin Mutant
The β-hairpin mutant contains two point mutations on the tip, K512A/H513A. Because
the mutant has WT-level ATPase activity in the absence of DNA, it is not suitable for
studying the ATP hydrolysis mode in the doping assay. However, because it has no
unwinding activity (Fig. 2.2D, 2.2E), this mutant can serve as the non-catalytic subunit in
the doping assay to study the subunit coordination for helicase activity. I performed the
doping assay using fork DNA and blunt-ended origin DNA. The helicase doping results
with this mutant revealed subunit coordination modes identical to those obtained from the
ATPase-defective tri-cis mutant, either using the fork DNA or origin DNA as the
unwinding substrates. Again, the change of helicase activity when doping with the β-
hairpin mutant fitted the semi-coordinated mode well (C=3, blue curve) with fork DNA
(Fig. 2.5A), but fitted the fully coordinated mode well (C=6, yellow curve) with origin
DNA (Fig. 2.5B). The matching results obtained from the β-hairpin mutant and the
ATPase mutant can be rationalized by the structural connection between the β-hairpin
and the ATP-pocket through the mechanical connector as shown in Fig. 2.1D, which
offers a structural basis for the functional coupling between the two distantly positioned
structural elements for helicase function.
27
Figure 2.5 Helicase doping study with LTag β-hairpin mutant containing
K512A/H513A mutations. The modeled curves for each possible coordination mode
(C=1, 2…6) for helicase activity are drawn as in Figure 2.4 in panels A-B. (A) In the
presence of fork-DNA, helicase doping result has the best fit for the C=3 “Trimer”
simulation or a semi-coordinated model (blue curve) (R
2
= 0.994). (B) In the presence of
a blunt-ended origin-DNA, helicase doping result fits an “Exponential” simulation curve
(R
2
=0.969). The error bars represent the standard deviation of three independent
experiments.
2.3 DISCUSSION
LTag Tri-cis Mutant as an Non-catalytic Subunit
Here I investigated the mechanisms of subunit coordination for ATP hydrolysis
and DNA unwinding by LTag hexameric helicase using mutant doping assay. The
construction of a catalytically “dead” mutant can provide a clean background for the
doping assay. In our efforts to obtain a mutant with no ATPase activity above
background, I generated single and double mutants of these three cis residues on the
ATP-pocket side, which always displayed some ATPase activity above background level
(Greenleaf et al., 2008). However, with triple mutations of the cis residues, the mutant
28
showed only background ATPase activity, thus successfully obtaining a fully ATPase-
inactive mutant for the doping assay. This tri-cis mutant also satisfies all the other
requirements for our doping assays, i.e. having WT-level hexamerization ability (with
itself or WT subunits), and retaining the ability for ATP to promote hexamerization (thus
the ability to bind ATP). These results are consistent with the prediction based on the
structure. The three mutated cis residues bond with the ATP through the apical water and
the Mg
++
at the ATP-pocket of its own subunit, but not with a neighboring subunit. Thus,
they are important for ATP hydrolysis, but not for inter-subunit interactions.
DNA-dependent Coordination for ATP Hydrolysis
The ring-shaped arrangement of the hexameric helicases indicates that six
subunits can coordinate with each other in some manner to hydrolyze NTP. Our data
showed that the subunit coordination mode for ATP hydrolysis in LTag hexamer greatly
depends on DNA substrates. In the absence of DNA, the proportional decrease of ATPase
activity in heterohexamers is consistent with a random or probabilistic mechanism of
ATP hydrolysis, similar to the previous reported case for ClpX in the absence of substrate
(Martin et al., 2005).
However, when ssDNA was present in the doping reaction, ATP hydrolysis is no
longer completely random, instead showing a semi-random mode that displayed a low
but consistent level of coordination (Fig. 2.3B). This behavior is different from T7 gp4
and SsoMCM. T7 gp4 switches dTTP hydrolysis from a random to a fully coordinated
mode when ssDNA is added to the reaction, i.e. all six sites are required for ATP
29
hydrolysis with ssDNA (Crampton et al., 2006). For the SsoMCM ATPase doping assay
where fork DNA instead of ssDNA was used, the same random mode was observed
with/without DNA (Moreau et al., 2007).
Unlike SsoMCM, when dsDNA (fork DNA or blunt-ended origin DNA) is
present in the ATPase doping assay of LTag, I observed a much more coordinated mode,
the semi-coordinated mode (C=3 model, Fig. 2.3C-D, Table 2.3). This C=3 model, or a
“trimer” model (see Experimental Procedures and Table 2.1) suggests ATP hydrolysis in
the presence of fork or origin DNA requires at least three WT subunits in a hexamer.
Because of the clear difference in coordination modes between ssDNA (semi-random
mode) and fork dsDNA substrate (semi-coordinated mode) for LTag helicase, I reasoned
that the process of unwinding of a fork DNA should be more than the pure translocation
on ssDNA by a LTag hexamer to displace another strand for LTag. On the other hand, T7
gp4 helicase showed full coordination in the presence of ssDNA, which implies that T7
gp4 may be able to unwind DNA by the pure translocation along ssDNA to displace the
other strand.
Taking the ATPase doping data together for LTag helicase, I observe a clear
DNA-dependent transition from random (C=1 linear curve) to semi-random (near C=2
curve) to semi-coordinated (C=3 curve) modes among the six ATP sites in the absence or
presence of different types of DNAs. The molecular explanation for this is unclear, and
perhaps a detailed study using single molecule approach may be needed to fully
understand the underlying principle. Nonetheless, these results demonstrate that the
30
subunit coordination for ATP hydrolysis of LTag hexamers operates differently from
other characterized hexameric motors.
Substrate-dependent Coordination For DNA Unwinding
As molecular motors, helicases couple the chemical energy derived from
nucleotide binding and hydrolysis to unwind duplex DNA. In the helicase doping assay, I
observed two distinct coordination modes when using fork DNA and a blunt-ended origin
DNA as unwinding substrates. For fork DNA unwinding, the helicase doping assay with
the ATPase-inactive tri-cis mutant revealed the C=3 semi-coordination mode (Fig. 2.4A),
matching that for the ATP hydrolysis in the presence of the same fork DNA (Fig. 2.3C).
This C=3 semi-coordination mode is a “Trimer” model that requires at least three WT
subunits in a hexamer (or can tolerate a maximum of three mutant subunits). However, I
cannot determine the specific arrangement of the three WT with the three mutant subunits
in a hexamer. Three possible arrangements are shown in Table 2.3 for mutant number
k=3: three consecutive WT subunits; two consecutive WT (or mutant) with one mutant
(or WT); alternating WT and mutant (like F1-ATPase type). The same trimer model for
both ATP hydrolysis and DNA unwinding using a fork DNA substrate is different from
the model previously described for SsoMCM. SsoMCM has a “pairs” coordination model
for unwinding a fork DNA, which requires a pair of active monomers to unwind fork
DNA, but uses a random mode for ATP hydrolysis even in the presence of fork DNA
(Moreau et al., 2007).
31
Interestingly, for origin DNA unwinding, a full coordination mechanism of LTag
subunits is apparent (Fig. 2.4B), indicating a switch from semi-coordination for ATP
hydrolysis (Fig. 2.3D) to a full coordination in DNA unwinding. This full coordination
mode for origin DNA unwinding suggests either a full sequential or a full concerted
mode among the six subunits, either of which would require all six subunits to be
functional. The mutant doping experiments cannot distinguish the two coordination
modes. However, the previous structural and biochemical evidence of LTag favor a
concerted mode. The crystal structures of LTag hexamers obtained so far are always in an
all-or-none nucleotide binding state, i.e. all empty, or all ADP, or all ATP in six
nucleotide pockets (Gai et al., 2004b; Huang et al., 1998; Li et al., 2003). Biochemical
analysis reveals 1:1 ratio of ATP or ADP binding to LTag subunits in solution (Huang et
al., 1998), which is consistent with an all-or-none nucleotide binding state. Even though
such an all-or-none mode observed for LTag favors the concerted mode for subunit
coordination, a sequential model cannot be excluded at this point. A more careful single-
molecule study using different unwinding substrates may be needed to resolve this issue
for LTag.
Since LTag functions both as initiator and helicase for viral DNA replication, one
possibility is that it may use different subunit coordination modes for different purposes
in different stages of replication. Fork DNA has ssDNA overhangs that allow LTag
hexamer to bind and translocate along the ssDNA, which can at least facilitate dsDNA
unwinding (Patel and Picha, 2000), while the blunt-ended origin DNA requires initial
melting of dsDNA before it matures into a fork-shaped substrate. During the initiation of
32
replication, LTag encircles two DNA strands at the origin to initiate melting and
unwinding. After a processive replication fork is formed, LTag may switch conformation
to encircle one strand of DNA for further unwinding. The number of DNA strands in the
central channel of LTag at these different stages may trigger different subunit
coordination modes for ATP hydrolysis and DNA unwinding. A switch of DNA stand in
helicase central channel has been proposed for eukaryotic DNA replication complex,
CMG, from replication initiation to unwinding (Fu et al., 2011).
Structural Connection and Functional Coupling between β-Hairpin and the ATP-
pocket
LTag hexamer has six β-hairpins protruding into the central channel, which is
conserved in many AAA+ helicases, and is involved in origin melting and DNA helicase
activity (Castella et al., 2006; Kumar et al., 2007; Liu et al., 2007; Shen et al., 2005). The
β-hairpin mutant has WT-level ATPase activity in the absence of DNA, but lacks the
DNA-dependent ATPase stimulation. The β-hairpin mutant did not show DNA binding
as WT in the absence of ATP, even though the mutant still showed DNA binding when
ATP was present (Shen et al., 2005), indicating proper DNA interactions via the β-hairpin
are important for ATPase stimulation. The result may reflect mechanical connection
between the β-hairpin and the ATP-pocket (Fig. 2.1D)(Gai et al., 2004b), which can
explain the coupling between ATP hydrolysis in the ATP-pocket to DNA
translocation/unwinding in the central channel.
33
Our helicase doping assay with the β-hairpin mutant showed the same two
coordination modes as the ATPase-inactive mutant (Fig. 2.5A, 2.5B, comparing with Fig.
2.4A, 2.4B). The same coordination modes obtained by using the two different types of
mutants further suggest the mechanical and functional coupling between the ATP-pocket
that binds/hydrolyses ATP and the β-hairpin tip that binds/translocates/remodels DNA.
The β-hairpin tip may play a sensor for the presence of substrate DNA in the central
channel, and convey the signal to the ATP-pocket via the structural connectors (Gai et al.,
2004b), leading to an stimulated ATP hydrolysis. This result is also consistent with the
structural observation that ATP binding, hydrolysis and release in the ATP-pocket
correlate with dramatically different β-hairpin conformations in the central channel
(Enemark and Joshua-Tor, 2006; Gai et al., 2004b).
In summary, I observed three distinct subunit coordination modes for ATP
hydrolysis in LTag hexamers, which can be switched from random to semi-random to
semi-coordinated, dependent not only on the presence/absence of DNA, but also on the
type of DNAs. For DNA unwinding, however, two different subunit coordination modes
were consistently observed using either an ATPase-defective mutant or a helicase-
defective β-hairpin mutant: a semi-coordinated mode when a fork DNA substrate was
used, and a fully coordinated mode when origin DNA was the substrate. These results
clearly indicate that the six subunits of LTag employ two distinctive coordination modes
for fork DNA unwinding and for origin DNA unwinding. This study provides evidence
that multiple working mechanisms of replicative helicases exist in the complicated
cellular replication process.
34
2.4 EXPERIMENTAL PROCEDURES
Materials
Oligonucleotides were obtained from Eurofins mwg/operon. The sequences of the
oligo nucleotides used are listed in Table 2.2. T4 polynucleotide kinase was purchased
from New England Biolabs. QuickChange
TM
Site-Directed Mutagenesis Kit was
purchased from Stratagene. Radiolabeled nucleotides were purchased from MP
Biomedicals. EnzChek Phosphate Assay Kit was purchased from Invitrogen. Glutathione
affinity column, Superdex 200 gel filtration column, Resource Q/S column, PGEX-6P-1
vector and PreScission protease were purchased from GE Healthcare.
Site-Directed Mutagenesis and Protein Purification
I performed site-directed mutagenesis using the QuickChange Site-Directed
Mutangenesis kit (Stratagene) in accordance with the manufacturer’s instructions. All
mutations were confirmed by sequencing the entire LTag coding sequence.
LTag131-627 wild type and mutant were produced by E.Coli expression system
as described previously (Li et al., 2003). Basically, the wild type or mutant protein was
expressed as a GST-LTag fusion using the PGEX-6P-1 vector, including a prescission
cleavage site between GST and LTag. Fusion protein was purified through a glutathione
affinity column, and then GST was cleaved by PreScission protease. The LTag protein
was eluted from the affinity column then further purified by ion exchange and Superdex
200 gel filtration chromatography. The protein was concentrated to 10mg/ml in a buffer
35
containing 25 mM Tris-Cl (pH8.0), 500 mM NaCl, and 10 mM DTT. All proteins were
quantified by using the Bradford method and Coomassie blue R-250 staining on SDS-
PAGE.
Oligomerization of LTag
The ability of LTag WT or mutant to form a hexamer was determined by
Superdex 200 gel filtration chromatography. 9 nmol of LTag WT or mutant protein in 1
ml of buffer containing 25 mM Tris-Cl (pH8.0), 250 mM NaCl, and with or without 1
mM ATP was incubated for 10 min at 18°C before loading to Superdex 200 column for
chromatography at 4°C. Hexamerization of LTag can be promoted by adding ATP alone
without the need of adding Mg
++
. The integration of absorbance data at 280 nm was used
to determine the total ratio of hexameric to monomeric peaks of LTag (Greenleaf et al.,
2008; Shen et al., 2005).
GST Pull-Down Assay
The ability of LTag mutant to associate with itself or WT subunits, as a surrogate
test for forming hexamers between mutant and WT, GST pull-down assay was carried out
by incubating 30 µl of GST-tagged WT LTag with non-tagged mutant (or GST-tagged
mutant with non-tagged wild type LTag) in a 1:1 molar ratio in the buffer of 25 mM Tris-
Cl (pH8.0), 500 mM NaCl for 10 min at 4°C. Then 30 µl of ATP solution containing 25
mM Tris-Cl (pH8.0), 4 mM ATP was added to original mixture and incubated for 1 hr at
4°C, to allow sufficient time for the protein mixture to form stable hexamers in the
presence of ATP (but no ATP hydrolysis as Mg
++
was not present). Next the mixture was
36
incubated with 15 µl of GST resin for 15 min at 4°C. The resin was washed extensively
with buffer containing 25 mM Tris-Cl (pH8.0), 250 mM NaCl and 2 mM ATP for three
times and analyzed for the proteins retained on the glutathione resin through association
with GST-tagged protein using 10% SDS-PAGE.
ATPase Assay
The ATPase assay was performed as described previously (Greenleaf et al.,
2008). The EnzChek phosphate assay kit was used according to the product manual
instructions. The ATPase assay was carried out in a reaction mixture containing 2 mM
ATP and 0.12 µM to 0.19 µM (as hexamer) of total LTag protein (either WT or mutant
alone or WT and mutant mixture) in a 50 µl buffer (10 mM MgCl
2
, 20 mM Tris-Cl
pH7.5) with or without the three types of DNAs (0.4 µM) shown in Table 2.2. After
incubating for 10 min at 37°C, 50 µl of stop solution containing 20 mM Tris-Cl pH7.5,
200 mM EDTA, 0.4 mM 2-amino-6-mercapto-7-methylpurine riboside (MESG) and
2U/ml Purine nucleoside phosphorylase (PNPase) was added to the reaction and
incubated for 30 min at 25°C. The free phosphate released by ATP hydrolysis interacts
with the MESG substrate and results in a MESG spectrophotometric absorbance shift
from 330 nm to 360 nm. The product absorbance was read by spectrophotomer (Bio-Rad)
at the wavelength of 360 nm. The data from assay were quantified and plotted by
statistical computing software R.
37
Helicase Assay
For the helicase assay, Y-shaped fork DNA with 44nt single-stranded DNA tails
and a 44nt duplex was obtained by annealing two oligonucleotides listed in Table 2.2.
The 146bp double stranded origin DNA substrate was prepared by PCR on the vector of
PGEX-2TK with a 64bp SV40 origin sequence insertion and the resulting sequence are
shown in Table 2.2. For the helicase assay, DNA substrate was 5’end-labeled with [γ-
32P] ATP by T4 polynucleotide kinase. Approximately 10 to 15 fmol of labeled fork or
origin DNA substrates was incubated with 0.04 µM (as hexamer for fork DNA) to 0.12
µM (as hexamer for origin DNA) of total LTag protein (either WT or mutant alone or
mixture of WT and mutant in different ratio) in helicase buffer containing 20 mM Tris-Cl
pH7.5, 10 mM MgCl
2
, 5 mM ATP, 1 mM DTT and 0.1 mg/ml bovine serum albumin for
45 min at 37°C. The reaction was terminated by adding to 0.1% SDS, 25 mM EDTA and
10% glycerol. The sample was analyzed on 12% native polyacrylamide gel in 1X Tris-
Borate-EDTA (TBE) buffer. The gel was dried and the radio-labeled oligonucleotide was
quantified by autoradiography. Data analysis was done by Quantity One and statistical
computing software R.
Description of Simulation of Doping Experiments
To provide the mechanistic detail about the coordination among six subunits of
LTag hexamer, a series of simulations were performed based on our models. Here I
assume: 1) LTag hydrolyzes ATP and unwinds DNA in the form of hexamers; 2) WT and
mutant subunits can associate with each other equally well to form hexamers; 3) each
38
ATPase-inactive mutant subunit in a hexamer can only affect one ATP site on one of its
interface, as the tri-cis mutant only contain mutations on one side of the subunit. The
formula of simulation is:
Y= (1)
In Eq. 1, p is the percentage of mutants, k is the number of mutant subunit in a
helicase ring, n is the number of subunits that forms a ring, for a hexameric ring, n=6, t
i
is
the probability of the ith arrangement in a certain combination of WT and mutant
heterohexamer, C
k
is the total number of conformations when there are k mutant subunits
in the hexamer; A
(m)
i
is the activity level [0,1] of the ith arrangement in model m. A
(m)
i
=1 if all subunits are wild type and A
(m)
i
= 0 if all subunits are mutant. Here mutant
means the monomer mutant, and arrangement means a specific permutation of six WT or
mutant subunits. Detailed deduction of activity in different models was presented in
Table 2.3. The models used in the paper are explained below.
Random/Probabilistic Model (C=1)
There is a linear relationship between measured activity and the percentage of
mutants (p). In a hexamer, each WT subunit contributes 1/6 of total activity; while each
mutant subunit has 0 activity (if all 6 subunits are WT, the activity is 1). This model
suggests that each subunit of the LTag hexamer is independent of each other during ATP
hydrolysis and unwinding. There is no cooperativity between subunits. The example of
39
this model is ClpX and ssoMCM for ATP hydrolysis (Martin et al., 2005; Moreau et al.,
2007). LTag ATP hydrolysis follows this model in the absence of DNA.
Fully Coordinated Model (C=6)
There is an exponential relationship between measured activity and the percentage
of mutants (p). Any, even one mutant subunit in the mixed protein hexamers would
abolish ATPase or helicase activity. This model suggests that all six subunits must be
catalytically active for ATP hydrolysis or helicase function. However, this model alone
cannot distinguish a fully sequential or a fully concerted coordinated mode for the
hexamer, as both will give identical curves with exponential decrease. An example of this
model is the T7 gp4 helicase, which is thought to operate in a sequential mode based on
other structural and biochemical data (Bird et al., 2000; Crampton et al., 2006; Donmez
and Patel, 2006; Enemark and Joshua-Tor, 2006; Singleton et al., 2000). The LTag
helicase mechanism follows this model in the presence of origin containing DNA
substrate.
Pairs Model (C=2)
This model has been suggested by the study of ssoMCM helicase mechanism
(Moreau et al., 2007). The relationship between measured activity and the percentage of
mutants is between a random/probabilistic model and a sequential/concerted model. This
model suggests that mixed hexamers could endure a few mutant subunits and still be
functional. Specifically, the pairs model indicates that the minimum requirement for
helicase activity of ssoMCM is to have two adjacent WT subunits.
40
Trimer Model (Semi-coordinated Model) (C=3)
This is another model between random/probabilistic and fully coordinated (or
sequential/concerted). This model suggests that the mixed hexamer requires three WT
subunits to be functional. In other words, the working set within one hexamer is a trimer,
any mutant monomer occurring within this trimer would stop the function of the
hexamer. The position of the trimer may have three possible arrangements as shows in
table 2.3. The LTag ATPase/helicase mechanism fits this model in the presence of fork
DNA.
Acknowledgments
I thank for the Nano-Biophysics core laboratory at USC for support. This work is support
in part by the WISE fellowship at USC, and by NIH R01 AI055926 to XSC.
Table 2.2 DNA Substrates Used in Doping ATPase and Helicase Assays
DNA
NAMES
DNA SEQUENCES
60nt ssDNA
5’-GAAGCCAATACAAAGGCTACATCCTCACTCGGGTGGACGGAAACGCAGAATTATGGTTAC
Fork DNA
a
5’-(dT)
44
GCTCGTGCAGACGTCGAGGTGAGGACGAGCTCCTCGTGACCACG
3’-(dT)
44
CGAGCACGTCTGCAGCTCCACTCCTGCTCGAGGAGCACTGGTGC
Blunt-ended
origin
DNA
b
5’- (AC…AT)
41
CACTACTTCTGGAATAGCTCAGAGGCCGAGGCGGCCTCGGCCTCTGCATAAATAAAAAAAATTA(GT….GA)
41
3’- (GT…AT)
41
GTGATGAAGACCTTATCGAGTCTCCGGCTCCGCCGGAGCCGGAGACGTATTTATTTTTTTTAAT(CA….CT)
41
a
: 44 dTs are not annealed;
b
: 64bp SV40 origin sequence is flanked by 41bp random DNA sequences.
!
41
Table 2.3 Deduction of activity in different models
Activity in different subunit arrangements A
(m)
i
k=0
𝐶
!
!
*(1-p)
6
t
i
= 1
Radom/Probabilistic
Model (C=1)
1
Pairs Model (C=2) 1
Trimer Model (C=3) 1
Sequential/Concerted
Model (C=6)
1
k=1
𝐶
!
!
*((1-p)
5
)*p t
i
= 1
Radom/Probabilistic
Model (C=1)
5/6
Pairs Model (C=2) 4/6
Trimer Model (C=3) 3/6
Sequential/Concerted
Model (C=6)
0
k=2
42
𝐶
!
!
*((1-p)
4
)*(p
2
) t
i
= 6/15 t
i
= 6/15 t
i
= 3/15
Radom/Probabilistic
Model (C=1)
4/6 4/6 4/6
Pairs Model (C=2) 3/6 2/6 2/6
Trimer Model (C=3) 2/6 1/6 0
Sequential/Concerted
Model (C=6)
0 0 0
k=3
𝐶
!
!
*((1-p)
3
)*(p
3
) t
i
= 6/20 t
i
= 12/20 t
i
= 2/20
Radom/Probabilistic
Model (C=1)
3/6 3/6 3/6
Pairs Model (C=2) 2/6 1/6 0
Trimer Model (C=3) 1/6 0 0
Sequential/Concerted
Model (C=6)
0 0 0
k=4
𝐶
!
!
*((1-p)
2
)*(p
4
) t
i
= 6/15 t
i
= 6/15 t
i
= 3/15
Radom/Probabilistic
Model (C=1)
2/6 2/6 2/6
Pairs Model (C=2) 1/6 0 0
Trimer Model (C=3) 0 0 0
Sequential/Concerted 0 0 0
43
Model (C=6)
k=5
𝐶
!
!
*(1-p)*(p
5
) t
i
= 1
Radom/Probabilistic
Model (C=1)
1/6
Pairs Model (C=2) 0
Trimer Model (C=3) 0
Sequential/Concerted
Model (C=6)
0
k=6
p
6
t
i
= 1
Radom/Probabilistic
Model (C=1)
0
Pairs Model (C=2) 0
Trimer Model (C=3) 0
Sequential/Concerted
Model (C=6)
0
Y= p is the percentage of mutants, k is the number of
mutant subunits in a helicase ring, n is the number of composite subunits in a helicase
ring. Here n=6 for our hexameric helicase, t
i
is the probability of the ith arrangements in a
certain combination of WT and mutant heterohexamer, C
k
is the total number of
arrangements when there are k mutant subunits in the hexamer; A
(m)
i
is the activity level
[0,1] of the ith arrangement in model m.
44
Chapter 3
Mechanisms of β-hairpin and F-loop Structures of
Simian Virus 40 Hexameric Helicase in DNA
Replication Initiation and DNA Unwinding
Reproduced with permission from Xian Jessica Yu, Bo Zhou, Dahai Gai, Stefan Vila,
Xiaojiang S. Chen 2012 Mechanisms of Involvement of the Residues of Simian Virus 40
Hexameric Helicase in Origin Melting and DNA Unwinding (Manuscript in preparation
for submission)
Author contributions: X.J.Y. purified proteins and DNA substrates, carried out DNA
binding assay, ATPase stimulation assay, helicase assay, biotin-streptavidin
displacement assay and potassium permanganate reactivity assay; B.Z. made mutant
constructs, purified proteins and carried out ATPase stimulation assay; S.V. assisted in
construct cloning and protein purification; D.G. provided advice for project design and
data analysis and X.S.C. supervised the project.
Simian Virus 40 (SV40) has been studied as the model system to elucidate the
mechanism of eukaryotic DNA replication. SV40 Large T Antigen (LTag) functions as a
replication initiator and a helicase in viral DNA replication. The β-hairpin and F-loop in
the central channel of LTag hexamer has been identified to play a key role in interacting
45
with DNA substrate. Here, I made a series of mutations on the tip of β-hairpin and an
adjacent F-loop structure. A variety of functional assays have been performed using these
mutants. I demonstrate that two neighboring β-hairpin tip residues (K512 and H513) and
residue F459 on F-loop are involved in DNA-dependent ATPase stimulation, in local
origin melting, ss- and ds- DNA translocation, and fork- and origin- DNA unwinding.
However, their relative functional roles in these activities are different, with K512 as a
sensor to recognize and convey the DNA signal from the central channel while H513 and
F459 as mechanics to interact with DNA base pairs for melting and unwinding. The
biochemical results support the structural observations that the positively charged residue
K512 interacts with DNA phosphate backbone through electrostatic interactions while
ring-shaped residues H513 and F459 interact with DNA by hydrophobic stacking.
3.1 Introduction
Simian Virus 40 (SV40) has been studied as the model system to elucidate the
mechanism of eukaryotic DNA replication (Fanning and Zhao, 2009; Kelly, 1988; Pipas,
2009). In eukaryotic cells, the initiation of DNA replication requires multiple protein
initiators to form a pre-replication complex (pre-RC) on replication origins that can
induce origin DNA melting and unwinding (Evrin et al., 2009; Fletcher et al., 2003; Lee
and Hurwitz, 2001; Remus et al., 2009). The process is highly complicated and precisely
regulated so that the mechanism is still not clear. However, in SV40 DNA replication,
SV40 Large T Antigen (LTag), which is a multi-functional protein, alone can fulfill the
46
viral DNA initiation job, including viral DNA origin recognition, origin melting,
replication fork unwinding, as well as recruitment of other essential cellular replicative
proteins (e.g. primase, polymerase, topoiseomerase) for further DNA elongation (Fanning
and Zhao, 2009). Studying the function of SV40 LTag provides a better understanding of
other eukaryotic DNA replication initiators and helicases.
SV40 LTag contains 708 amino acids and folds into three major functional
domains: a Dna-J homology domain (JD), an origin binding domain (OBD), and a
helicase domain (Fig. 3.1A) (Borowiec et al., 1990; Simmons, 2000). LTag recognizes
the origin through sequence-specific interactions via OBD domain and assembles into a
double hexamer at the origin (Cuesta et al., 2010). LTag melts the origin and
unwinds/translocates on DNA substrate majorly through helicase domain. The helicase
domain belongs to both AAA+ (ATPase associated with various cellular activities)
family and helicase superfamily III. The active form of LTag helicase is a ring-shaped
hexamer formed by six identical monomers. There are six ATP binding pockets between
adjacent monomers. Like other AAA+ family members, LTag couples the energy
produced by ATP hydrolysis to its DNA remolding work.
To achieve efficient unwinding, LTag requires a number of activities as showed
in a pyramid flowchart (Fig. 3.1C). LTag hexamerization is promoted through ATP
binding. Hexamer formation and ATP binding occur during LTag hexamer assembly on
origin DNA. Similarly, DNA interactions on LTag stimulate the ATPase activity and the
energy is transferred to further unwinding/translocation work of the hexamer. The
47
hexamer assembly on DNA origin also triggers local DNA structure change and causes
base pairs melting in the presence of ATP binding (Borowiec and Hurwitz, 1988).
Therefore, the interaction of LTag with DNA substrate involves multiple activities,
including ATPase stimulation, local origin melting, and eventually DNA strand
separation.
The co-crystal structure of LTag helicase domain with various nucleotide-bound
states has revealed a significant conformational change of LTag helicase domain,
including a longitudinal movement of six conserved β-hairpin and DR/F loop structures
along the central channel surface (Gai et al., 2004b) (Fig. 3.1B). In addition, mutagenesis
studies have identified that residue K512, K516 and H513 on β-hairpin tip and residue
F459 on an adjacent loop (F-loop) are critical in DNA binding and helicase activity
(Kumar et al., 2007; Shen et al., 2005). The recent co-crystal structure of LTag with
origin DNA also suggested an important role of these tip residues in origin DNA melting
(Chang et al., 2012). However, the molecular mechanism of these residues in interacting
with DNA substrates at different stages of replication initiation, i.e. ATPase stimulation,
origin melting, DNA unwinding, and ssDNA/dsDNA translocation, remains unsolved.
To understand the order and the mechanism of the β-hairpin and the F-loop
structures in these multiple activities, I constructed a series of mutations with different
side chain properties on the two critical β-hairpin tip residues K512, H513 and one F-
loop structure residue F459 in the central channel. I performed functional activity assays
for these mutants, including ATPase stimulation, origin and fork-shaped DNA
48
unwinding, ssDNA and dsDNA translocation and local origin DNA melting. The results
demonstrated the critical roles of the central channel β-hairpin and loop structures in
DNA replication initiation.
Table 3.1. Residues used to substitute the β-hairpin and F-loop residues
Residues Mutated Residues
K512 G A L R E
H513 G A V L I M F W Y
F459 A L I D M H W Y
K512, H513 HV RV WV HK
49
Figure 3.1 SV40 LTag domain structure and the various activities. (A) The linear
SV40 LTag domain structure. (B) The superposition of LTag helicase domain structures
in ATP-bound, ADP-bound, and nucleotide (nt)-free states, all without DNA bound.
Major structural shifts of the β-hairpins (β-hp) and the DR/F loops along the central
channel from the C-to-N direction (indicated by the arrows) occur when the nt-free state
binds ATP. The red arrows indicate the transition of the β-hp from the nt-free (yellow β-
hp) to the ATP-bound state (pink β-hp), and the blue arrows indicate the DR/F loop shift
from the nt-free (yellow loop) to the ATP-bound state (pink loop) (unpublished paper
data). (C) Activities of LTag in DNA replication initiation. The pyramid shows the
hierarchy and the interdependence of the various activities of LTag.
50
3.2 Results
Mutant Protein Purification
The LTag construct used for this study contains residues 131-627 (termed
LTag131 hereafter) that includes OBD and the helicase domain (Zn and AAA+ domain).
LTag131 is capable of recognizing and melting origin DNA (Yu et al., 2012).This
construct purified from E. coli has the similar activity as the full-length LTag purified
from insect cells in unwinding origin and fork DNA in vitro (Gai et al., 2004a). In this
report, I constructed a series of mutations based on LTag131 to characterize the specific
functional roles of three central channel residues in origin melting and DNA unwinding.
The three residues under study are a positively charged residue K512 and two ring-
shaped residues H513 and F459, as these residues were shown to play important role for
helicase function, but how these residues are involved in unwinding is unclear. To
understand if these residues are involved in origin melting, in DNA translocation, or
unwinding, they were mutated individually or in combination to a series of different
residues that cover a range of side chain size, charge and shape (Table. 3.1). LTag Wild
Type (WT) and mutant proteins were expressed and purified from E. coli, and protein
concentration were quantified by using Bradford method and calibrated using Coomassie
blue staining method on SDS/PAGE.
Hexamerization and DNA Binding
I examined the ability to hexamerize for the mutants containing various
substitutions on residue K512, H513 and F459, by gel filtration, and showed that all the
51
amino acid substitutions at these three positions maintained the same level of
hexamerization, indicating these mutants has the same structural integrity as the WT
LTag.
I next examined the protein-DNA interaction of these mutants. The previous
mutagenesis study showed that all the mutants of K512, H513 and F459 bind to both
ssDNA and dsDNA in the presence of ATP. ATP is required for the binding (Shen et al.,
2005). The explanation is that ATP triggers the hexamerization of LTag, which in turn
facilitates the LTag assembling on DNA substrate as a hexamer or double hexamer. In
addition, recent co-structure of LTag with origin DNA has showed three contacting
regions with DNA: Zn domain, β-hairpin and F-loop regions (Chang et al., 2012) (Fig.
3.1B). Interactions between the Zn domain and dsDNA are mainly through two DNA
phosphate backbones by positively charged residues on this region. The Zn domain also
anchors LTag to the replication origin during assembly and possibly provides an
anchoring point that allows the AAA+ domain to rotate relative to DNA in order to melt
and unwind DNA (Chang et al., 2012). Therefore, even when the residue on β-hairpin or
DR/F loop regions was mutated, the mutant LTag still can bind DNA and form hexamer
with the Zn domain, but other functions such as unwinding may be affected. To further
confirm the mutant-DNA binding ability, a binding test was performed with double
mutant K512A/H513A in the presence of ATP-γ -S. As seen in Fig. 3.2A and 3.2B,
double mutant K512/H513 bound to both ssDNA and dsDNA, which is only about 50%
less binding compared with WT LTag (Fig. 3.2C). Base on the structure and biochemical
52
results, all of the mutants tested in this report can bind ssDNA and dsDNA in the
presence of ATP or ATP analogs.
Figure 3.2 DNA binding anisotropy of LTag WT and mutants. (A) ssDNA binding
curves for LTag WT, K512/H513 and Tri-cis mutants in the presence of ATP-γ-S. (B)
dsDNA binding curves for LTag WT, K512/H513 and Tri-cis mutants in the presence of
ATP-γ-S. (C) Table of binding results on both ssDNA and dsDNA. The apparent values
of K
d
(K
d
,
app
) for ss- and dsDNA binding by WT and mutated LTags in the presence of
ATP-γ-S, measured by rotational fluorescence anisotropy, were calculated by fitting the
resultant binding curve to equation (1). Errors in Hill coefficients (n) were typically less
than 10%.
53
ATPase Stimulation by ssDNA and dsDNA
The ATP hydrolysis of WT LTag can be stimulated by DNA. I conducted the
ATPase stimulation assay for all mutants (Fig. 3.3). The result was expressed as the
percentage (%) of the WT in stimulated ATPase activity. Among the five K512 mutants,
all but one have completely lost the ATPase stimulation with either ssDNA or dsDNA
(Fig. 3.3A). K512R is the only mutant that showed about 30% of the WT activity on
dsDNA.
Interestingly, the DNA stimulated ATP hydrolysis showed a different profile for
the nine H513 mutants (Fig. 3.3B). In the presence of ssDNA, seven out of the nine
mutants retained ATPase stimulation ability (Fig. 3.3B). The two mutants that lost the
activity have branched side chain, Val and Ile at H513. All substitutions by large and
long hydrophobic and ring-shaped side chain, Leu, Met, Phe, and Tyr, retained close to
WT activity. The substitution by small side chains, Ala and Gly, showed less than 50%
WT activity. When dsDNA was present, however, the result was somewhat opposite.
The substitution by smaller side chains, Ala and Gly, had 2-3 times higher activity than
WT (Fig. 3.3B). Substitution with long aliphatic side chains, Leu and Met, had higher
activity than the ring-shaped side chains. The common result for both ssDNA and dsDNA
stimulation is the mutant with substitution by Val and Ile, both had no stimulation
activity.
These results suggest that both K512 and H513 are important in sensing the
presence of DNA in the channel and relay the signals to the ATP pocket. However, K512
54
is apparently more critical in this signaling process, and thus more sensitive to mutational
substitution. In other words, H513 is less critical in this signaling process because most of
the substitutions at this position didn’t abolish the stimulated ATPase activity. To further
test this hypothesis, I made some double mutants to identify the roles of these two
residues in substrate-dependent ATPase stimulation. I either switched the two residues or
made K512 hydrophobic or with positive charge but kept H513 inactive in ATPase
stimulation by replacing both residues with HK, HV, RV, or WV. In Fig. 3.3C, all the
double mutants abolished stimulation activity in the presence of ssDNA, and retained
very little stimulation activity in the presence of dsDNA, which means that the roles of
K512 and H513 are different in sensing the DNA and the two positions are non-
interchangeable.
The Channel loop residue F459 mutants showed ATPase stimulation profile
similar to H513 mutants (Fig. 3.3D). Among all the F459 mutants, only F459W and
F459Y showed ssDNA-stimulated ATPase activity (117% and 66%, respectively). Some
small side chain substitutions (L, I, D, M) even showed inhibited ATPase activity, which
means that the mutants negatively affected the basal ATPase activity. However, in the
presence of dsDNA, all of the mutants except one stimulated ATPase activity. As was the
case with ssDNA, F459W and F459Y showed the most stimulation (about 121% and
280%, respectively) with dsDNA. The results suggest that F459 is important, but less
critical than K512 for DNA-stimulation of ATP hydrolysis.
55
Figure 3.3 ATPase stimulation assay in the presence of ssDNA or dsDNA. (A) - (B)
ATPase stimulation assay with LTag K512 mutants (A), LTag H513 mutants (B), LTag
K512/H513 double mutants (C), and LTag F459 mutants (D). The stimulated ATPase
activity of LTag WT was normalized to 100%, the mutants’ stimulated activities were
calculated as the percentage of WT activity. The error bars in all panels represent the
standard deviation of three independent experiments.
Helicase Activity on Fork-Shaped DNA and Origin dsDNA
The helicase activity of the mutants was determined by displacing a
32
P-labeled
ssDNA from dsDNA. Two substrates were used for helicase assay, the fork-shaped DNA
and the origin-containing blunt end dsDNA. The unwinding of the origin DNA represents
the whole replication initiation process and it requires LTag to recognize the origin, melt
56
the origin and propagate to further strand separation.
In the fork DNA helicase assay, the unwinding activity of all the K512 mutants
and the K512/H513 double mutants were completely abolished (Fig. 3.4A and 3.4B).
Most of the H513 and F459 mutants were inactive in fork unwinding except H513M,
H513F, H513W, H513Y and F459W, showing 42%, 104%, 95%, 100% and 33.2%,
respectively comparing to the WT (Fig. 3.4C). In the origin DNA unwinding assay, the
mutants’ activity profile was highly similar as the unwinding on the fork DNA. K512 and
K512/H513 double mutants were all inactive in unwinding origin DNA (Fig. 3.5A and
3.5B). H513M, H513F, H513W, H513Y and F459W consistently showed unwinding
activity as 49%, 136%, 32%, 87% and 35%, respectively comparing to the WT 100%
(Fig. 3.5C). These results suggest that K512, H513 and F459 are all important in both
fork and origin DNA unwinding through the direct interactions with DNA. Substitutions
of H513 by ring shaped residue or long aliphatic side chain would rescue the helicase
activity on both DNAs. Substitutions of F459 with ring-shaped hydrophobic residue can
also retain the helicase activity on both DNAs.
57
Figure 3.4 Unwinding activity of LTag mutants on fork-shaped DNA. B: Boiled
DNA; UB: unboiled DNA with no protein. The protein concentration used in the assay
was 0.45µM as monomer. The mutant’s activity was expressed as the percentage (%) of
WT activity (100%) in each bar chart. (A) Helicase assay of various mutants of K512.
(B) Helicase assay of various mutants of K512/H513. (C) Helicase assay of various
mutants of K513 and F459.
58
Figure 3.5 Unwinding activity of LTag mutants on origin dsDNA. B: Boiled DNA;
UB: unboiled DNA with no protein. The protein concentration used in the assay was
0.45µM as monomer. The mutant’s activity was expressed as the percentage (%) of WT
activity (100%) in each bar chart. (A) Helicase assay of various mutants of K512. (B)
Helicase assay of various mutants of K512/H513. (C) Helicase assay of various mutants
of K513 and F459.
Translocation on ssDNA or dsDNA
Some of H513 mutants (e.g. H513G, H513A, H513L) and F459 mutants (e.g. F459L,
F459Y) exhibited certain levels of stimulated ATPase by ssDNA and/or dsDNA (Fig.
3.3), but their fork or origin unwinding activity were totally lost (Fig. 3.4C, lanes 4-6, 17,
26 and 3.5C, lanes 4-6, 17, 26). The energy from the stimulated ATP hydrolysis may be
coupled for translocation on DNA, but not coupled to DNA unwinding. Alternatively,
59
simply binding DNA can stimulate repeated cycle of ATP hydrolysis similar to the
stalled or slippage situation. To test if the DNA-stimulated ATP hydrolysis of these
mutants without coupling to DNA unwinding can be coupled to translocation on ssDNA
and dsDNA, I performed a translocation assay in which displacement of
biotin/streptavidin (Bio/SA) block on ssDNA or dsDNA was examined as described in
(Morris et al., 2001). As shown in Fig. 3.6, LTag WT cannot displace the Bio/SA block
in the presence of ATP-γ –S (lanes 2-4), but displaced the block in the presence of ATP
(lanes 5-7). All of the K512 mutants cannot displace the Bio/SA block (Fig. 3.6A, lanes
8-17), but four of the H513 mutants (H513M, H513F, H513W and H513Y) and F459W,
all of which have displaced the Bio/SA block on ssDNA (Fig. 3.6B, lanes 18-25 and 3.6C,
lanes 20, 21).
For dsDNA translocation, it is important to test the protein concentration at which
LTag can translocate on dsDNA but does not unwind the DNA. The mutant translocation
profile on dsDNA was highly similar to that on ssDNA. Those mutants that cannot
displace Bio/SA block on ssDNA still couldn’t displace it on dsDNA, and those other
mutants that can displace the protein block on ssDNA consistently displaced it on dsDNA
(Fig. 3.7B, lanes 18-25 and 3.7C, lanes 20-21). Interestingly, H513 mutants G, A, L and
F459 mutants L and Y that didn’t show any translocation activities on both DNAs even
they stimulate ATPase activity on both DNAs. This suggests that the energy produced by
these mutants is not coupled to either translocation or unwinding. Furthermore, by
comparing the translocation data with the unwinding data (Fig. 3.4C, lanes 12-15 and 25;
Fig. 3.5C, lanes 12-15 and 25; Fig. 3.6B, lanes 18-25; Fig. 3.6C lanes 20-21; Fig. 3.7B
60
lanes 18-25; Fig. 3.7C, lanes 20-21), it is apparent that a ring-shaped residue on 513 and
459 plays an important role in translocation on ssDNA/dsDNA as well as in DNA
unwinding.
Figure 3.6 Helicase-mediated biotin-streptavidin displacement assay on ssDNA.
ssDNA: free DNA with biotin; ssDNA-SA Cplx: Complex of DNA and Streptavidin; WT
(ATP-γ-S): biotin-streptavidin displacement assay measured with LTag WT in the
presence of ATP-γ-S; WT (ATP): biotin-streptavidin displacement assay measured with
LTag WT in the presence of ATP. Protein concentration tested for WT and mutants in the
assay was: 0.09 and 0.18µM. (A) Biotin-streptavidin displacement activity of various
K512 mutants. (B) Biotin-streptavidin displacement activity of various K513 mutants.
(C) Biotin-streptavidin displacement activity of various F459 mutants.
61
Figure 3.7 Helicase-mediated biotin-streptavidin displacement assay on dsDNA.
dsDNA: free DNA with biotin; dsDNA-SA Cplx: Complex of DNA and Streptavidin;
WT (ATP-γ-S): biotin-streptavidin displacement assay measured with LTag WT in the
presence of ATP-γ-S; WT (ATP): biotin-streptavidin displacement assay measured with
LTag WT in the presence of ATP. Protein concentration tested for WT and mutants in the
assay was: 0.18 and 0.36µM. (A) Biotin-streptavidin displacement activity of various
K512 mutants. (B) Biotin-streptavidin displacement activity of various K513 mutants.
(C) Biotin-streptavidin displacement activity of various F459 mutants.
Local Origin Melting Detected by Potassium Permanganate Reactivity Assay
The recent co-crystal structure of LTag131 with origin DNA has demonstrated the
binding of origin DNA in the central channel through residues include K512, H513 and
F459 in the central channel side (Chang et al., 2012). To confirm the important function
62
of these residues in local origin DNA melting, a potassium permanganate (KMnO
4
)
reactivity assay was performed to detect distorted DNA caused by LTag binding. A top
strand labeled 100bp origin DNA (Fig. 3.8A) was incubated with LTag WT or mutant in
the presence of ADP and treated with KMnO
4
for 1 min at 37°C. If there is a melted
region, the thymidine residues will be oxidized by KMnO
4
, and the oxidized thymidine
position can be cleaved by piperidine at 90°C. I performed the KMnO
4
reactivity assay,
and denaturing Urea-PAGE gel electrophoresis was used to analyze the melting by
detecting cleaved DNA fragments. As seen in Fig. 3.8B and 3.8C, WT LTag generated a
few cleaved bands at the lower part of the gel, which were not present in the absent of
LTag protein (lane UB in Fig. 3.8B). For the most part, the cleaved bands were only
present for the mutant LTag with origin unwinding activity, but not present for those
mutant without helicase activity. Interestingly, H513L, which is active in ATPase
stimulation but not in unwinding and translocation, showed the cleavage band indicative
of local origin melting activity. Because ATP hydrolysis is not required in this assay and
local origin melting is caused by LTag-DNA binding, it appears that small change of the
interactions between LTag side chains and DNA caused by changing the side chain
geometry even in a subtle way can affect melting of the origin. These results clearly
indicate that all the three residues on the β-hairpin and the channel loop, K512, H513 and
F459, are playing a critical role in origin DNA melting.
63
Figure 3.8 Potassium permanganate reactivity assay. (A) A sequence of 100bp
dsDNA containing the replication origin of SV40. The four pentanucleotides GAGGC
(PEN in cyan) are flanked by the AT rich sequences (AT in yellow) and the early
palindrome sequence (EP in magenta). The black dots on the top of each Thymine (T)
residue indicate the potential KMnO
4
attacking positions. The asterisk symbol above the
brackets shows the modified Ts in the sequence. (B) The potassium permanganate
reactivity assay for H513 mutants. (C) The potassium permanganate reactivity assay for
F459 and K512 mutants. UB: unboiled DNA with no protein. B: Boiled DNA. The
asterisk symbol shows the modified Ts in the permanganate reactivity assay, which
locates outside of the origin sequence but close to AT region.
64
3.3 Discussion
As a DNA replication initiator and helicase, SV40 LTag participates in a series of
activities with DNA once assembles onto the origin. The channel β-hairpin and F-loop
are two structural elements directly contacting DNA (Chang et al., 2012). The possible
functions of the three residues on the β-hairpin and F-loop structures have been
investigated through extensive mutational substitutions and biochemical studies. The
results showed that these residues are all important, but playing different roles in the
DNA-dependent ATPase stimulation, local origin melting, DNA translocation and
unwinding (Fig. 3.9).
K512, not H513, Is Required in ATPase Stimulation Induced by DNA Substrate
LTag ATP hydrolysis happens in the ATP-binding pocket, which is between two
adjacent monomers and is distal from the central channel. Structural and biochemical
study of LTag ATP hydrolysis mechanism revealed the mechanical and functional
coupling between the ATP-pocket that binds/hydrolyses ATP and the β-hairpin tip that
binds/translocates/remodels DNA (Gai et al., 2004b; Yu et al., 2012).
There are two residues, K512/H513, on the β-hairpin tip that contact DNA. Our
results indicate that the importance of these two residues for DNA-dependent ATPase
activity differs. The β-hairpin tip residue K512 is more critical for the DNA-dependent
ATPase stimulation activity because all substitutions at this position showed completely
abolished the ssDNA-dependent stimulation, and all but one lost the dsDNA-dependent
activity (Fig. 3.3A, 3.3C). Compared with K512 mutations, most H513 substitutions
65
Figure 3.9 Activity summary of mutations on β-hairpin and F-loop structures. Each
bar chart shows four activities of the mutant with different colors, including ssDNA-
(blue) and dsDNA- (red) dependent ATPase stimulation activities, fork-DNA (green) and
origin DNA (purple) unwinding activities. On the bottom of each bar chart displays a
table of DNA translocation activity on ssDNA and dsDNA. Y means positive activity and
N means negative activity in DNA translocation.
66
(seven out of nine) retained ssDNA-dependent stimulation and five out of nine H513
substitutions retained dsDNA-dependent stimulation (Fig. 3.3B). These results suggest
that H513 can be substituted with various side-chains and still possess DNA-dependent
stimulation of ATPase activity, thus plays a relatively less critical role in this process.
The structure shows that K512 interact with the phosphate backbones of DNA, while the
side chain of H513 can interact with DNA bases (Chang et al., 2012). Based on the
different results of K512 and H513 in ATPase stimulation, it suggests that sensing the
existence of DNA substrate in the LTag central channel may due to the charge-charge
interaction between the negatively charged DNA phosphate backbone and the Lys
residue on β-hairpin. Interestingly, Lys to Arg mutation essentially abolish the DNA-
dependent stimulation, indicating Arg cannot fulfill the functional role of Lys, possibly
because Arg tends to insert into the minor groove of dsDNA or interact with bases of
ssDNA (Rohs et al., 2009). Therefore, the positive charge of side chain alone at K512
position is not sufficient, and there is a very particular requirement for a Lys residue at
this position for the DNA-dependent ATPase stimulation.
Origin Melting Activity of LTag Mutants
The assembly of SV40 LTag hexamer/double hexamer on origin DNA alone can
induce origin melting (Borowiec and Hurwitz, 1988; Mastrangelo et al., 1989). The
biochemical study of local origin melting using circular plasmid DNA showed the
KMnO4 modified melted region at both AT and EP regions of the origin sequence
(Borowiec and Hurwitz, 1988). Here, by using shorter linear origin DNA (100bp), I
67
showed that the KMnO4 modification pattern was focused on the region next to the AT
region but outside the core origin sequence (Fig. 3.8B and 3.8C, lane 3). In our melting
assay, I showed that single Ala substitution on K512, H513, or F459 all displayed no
origin melting activity (Fig. 3.8B, lane 5 and Fig. 3.8C, lane 4, 9), indicating all these
three residues are required for origin melting. Furthermore, the substitution of ring-
shaped side chain (H513F, Y and F459W) or long aliphatic side chain (H513L) can retain
the origin melting activity. The results suggest that during local origin melting or
distortion, the hydrophobic packing interactions of H513 and F459 with DNA are
important (Fig. 3.8B, lane 6, 8, 9 and Fig. 3.8C, lane 5). For K512, all of the
substitutions, including Arg substitution, lost the melting activity, suggesting the way
K512 binds DNA are critically important for origin melting.
LTag Helicase Activity
LTag couples the energy produced by ATP hydrolysis to unwind duplex DNA.
The helicase function of β-hairpin tip residues K512, H513 and F-loop residue F459 have
been determined by detecting the displacement of a single oligo annealed on the M13
single stranded plasmid (Shen et al., 2005). In this report, I systematically tested the
helicase function of LTag with two different substrates, i.e. fork DNA and origin DNA.
Fork-shaped substrate mimics the replication fork after origin melting, while unwinding
origin dsDNA represents the whole replication initiation process. LTag can efficiently
unwind both of them (Fig. 3.4A and 3.5A, lane 3). The substitution of K512, H513 and
F459 with alanine totally abolished the unwinding activity on both substrates (Fig. 3.4A
68
and 3.4C, bar charts). Some of other substitutions on these three residues showed helicase
activity, and the results on fork DNA and origin DNA are largely the same (Fig. 3.4 and
Fig. 3.5, bar charts). Combined with the ATPase stimulation results in Fig. 3.3, it clearly
suggests that the defect of K512 mutants in unwinding activity correlate well with their
loss of DNA-dependent stimulation of ATPase activity. However, for H513 and F459
mutants that still retained the DNA-dependent ATPase stimulation, the substituted
residues were ring-shaped or long aliphatic side chains and these mutants also possess the
unwinding activity (Fig. 3.4 and Fig. 3.5, bar charts).
LTag Translocates on both ssDNA and dsDNA
One of the prevailing models of duplex unwinding of hexameric helicases were
based on studies of ring helicases, which suggests that the helicase binds and translocates
unidirectionally on ssDNA, displacing the other strand (Donmez and Patel, 2006;
Galletto et al., 2004; Hacker and Johnson, 1997; Kaplan, 2000; Kaplan et al., 2003;
Kaplan and Steitz, 1999; Shin et al., 2003; Yu et al., 1996) In this “steric exclusion”
model, unwinding and translocation is functionally coupled. In addition, other helicases,
e.g. RuvB, can translocate on dsDNA without unwinding the duplex, which shows the
uncoupling of the two activities (Putnam et al., 2001; West, 1996; Yamada et al., 2001).
For SV40 LTag, I show here that it can translocate not only on ssDNA, but also on
dsDNA without unwinding. The mutants investigated in this study show a correlation of
ssDNA translocation activity with unwinding activity on fork DNA substrate, suggesting
that LTag unwinds fork DNA substrate through “steric exclusion” model that encircles
69
one strand and excludes the other one. The translocation and unwinding are functionally
coupled (Fig. 3.4 and Fig. 3.6). However, our dsDNA translocation results indicate that
LTag translocation can occur without unwinding DNA at lower protein concentration
(Fig. 3.7A, 3.7B and 3.7C, lane 6, 7), which suggests that these two functions can be
uncoupled. In addition, these results show that K512, H513 and F459 residues are all
required in translocation on ssDNA and dsDNA.
The Role of the Residues on the β-hairpin and Loop Structure
The three residues studied here locate on the β-hairpin tip and F-loop structure of
LTag central channel, which forms the narrowest bottleneck to interact with DNA (Fig.
1B). The functional studies in this report firstly identified the different roles of the two
neighboring residues (K512 and H513) on the β-hairpin of LTag. K512 is required for
DNA-dependent ATPase stimulation, which suggests a sensor role of this Lys residue.
The further unwinding and translocation activities are both dependent on the energy
produced from the ATP hydrolysis. Therefore, the defects of K512 mutants in these
activities may be due to reduced ATP hydrolysis. In addition, K512 is required for local
origin melting, which is required of ATP binding but not ATP hydrolysis (Borowiec and
Hurwitz, 1988). From the results, I suggest that K512 could interact with DNA phosphate
backbone through charge-charge interactions, which is in agreement with the previous
work and recent structural data (Chang et al., 2012; SenGupta and Borowiec, 1992,
1994). Unlike K512, a variety of substitutions of H513 on β-hairpin and F459 on F-loop
structure supported ATPase stimulation, which indicates that H513 and F459 are less
70
critical in sensing the DNA and transferring the signal to ATP-binding pocket. However,
H513 and F459 are both required in all other activities, such as origin melting, DNA
unwinding and translocation. However, hydrophobic ring-shaped substitutions of H513
(W, F and Y) and F459 (W) recovered these activities. It suggests that H513 and F459
could interact with DNA through hydrophobic stacking with base pairs, which is also
consistent with the structural observation that H513 inserts into the minor groove of DNA
(Chang et al., 2012) (Fig. 3.10).
The β-hairpin is highly conserved structure for many ring hexameric helicases
(Fletcher et al., 2003; Liu et al., 2007; Satapathy et al., 2010). The Lys and His on the β-
hairpin at the position corresponding to K512 and H513 in SV40 LTag are also conserved
in papillomavirus E1 helicase, both belongs to the SF3 family member of helicase. The
Lys residue (K506) on the E1 β-hairpin is reported to be required for both local origin
melting and DNA helicase function, and the His residue (H507) on E1 β-hairpin is only
required for local origin melting, but not for unwinding (Liu et al., 2007). The reason for
the different role of the equivalent β-hairpin His residue of these two closely related
helicases is not clear. One possibility could be due to the different structural arrangement
of the β-hairpins in the central channel, i.e. a planar β-hairpin of LTag vs. a staircase-
arranged β-hairpin of E1 (Enemark and Joshua-Tor, 2006; Gai et al., 2010; Gai et al.,
2004b).
71
Fig. 3.10 Interactions of residues on β-hairpin and loop structures with EP-origin
DNA (A) The interactions of residues with DNA backbone and bases on one subunit. (B)
The interactions of residues with DNA backbone and bases on the other subunit. Both
panels show that K512 interacts with DNA phosphate backbone through charge-charge
interactions while H513 interacts with DNA base pairs through hydrophobic stacking
(Chang et al., 2012).
A Model for SV40 Viral DNA Replication Initiation and DNA Unwinding
Based on systematic mutagenesis study and available structural data, I propose a
model for LTag DNA replication initiation and DNA unwinding. Once LTag assembles
on the origin. The residue K512 starts to interact with DNA phosphate backbone through
electrostatic interaction. There are two consequences from the interaction: one is that the
binding induces the local structure distortion or melting at EP or AT region; the other one
is the transferring of the DNA signal from central channel to ATP-binding pocket
through structure connection resulting in stimulated ATP hydrolysis. Meanwhile, residue
H513 and F459 interact with DNA base pairs through hydrophobic stacking. During the
melting stage, the hydrophobic stacking with bases could stabilize melted bases and
72
prevent it from re-annealing. Therefore, each residue has its own responsibility to
coordinate the initiation job. Although the narrowest bottleneck formed by β-hairpin and
DR/F loop is not big enough to hold normal B-formed dsDNA, the interactions between
DNA and these residues actually could squeeze two strands to fit the channel by
interrupting the base pairs.
The β-hairpin and F-loop have been shown to move longitudinally along the
central channel in large distance upon ATP binding, hydrolysis and release (Gai et al.,
2004b). The ATP-triggered movement is proposed for translocation on DNA and
unwinding dsDNA. For DNA unwinding, ring-shaped residues H513 and F459 could
interact with dsDNA through the same mechanism as they do for melting through
hydrophobic stacking with bases. In addition, the these two residues may form a working
line to pull the dsDNA into the center for unwinding and the resulted ssDNA could exit
through the side channel proposed in the looping model (Gai et al., 2004b; Li et al.,
2003). Therefore, two ring-shaped residues (H513 and F459) are required to maintain the
dsDNA destabilizing state during the strand movement trigged by ATP hydrolysis in the
central channel (Fig. 3.10).
In summary, the biochemical study of residues on β-hairpin and loop structure
supports the essential role of these residues in multiple activities involved in DNA
replication initiation and DNA unwinding. I demonstrate that two neighboring β-hairpin
tip residues (K512 and H513) and residue F459 on F-loop are involved in DNA-
dependent ATPase stimulation, in local origin melting, ss- and ds- DNA translocation,
73
and fork- and origin- DNA unwinding. However, their relative functional roles in these
activities are different, with K512 as a sensor to recognize and convey the DNA signal
from the central channel while H513 and F459 as mechanics to interact with DNA base
pairs for melting and unwinding. The biochemical results support the structural
observations that the positively charged residue K512 interacts with DNA phosphate
backbone through electrostatic interactions while ring-shaped residues H513 and F459
interact with DNA by hydrophobic stacking (Chang et al., 2012). The role and the
mechanism of residues on the β-hairpin tip and loop structure provides the insight onto
other hexameric helicases and better understanding of DNA replication initiation in
eukaryotic cells.
3.4 Experimental Procedures
Materials
Oligonucleotides were obtained from Eurofins mwg/operon. The sequences of the
oligo nucleotides used are listed in Table 3.2. T4 polynucleotide kinase was purchased
from New England Biolabs. QuickChange
TM
Site-Directed Mutagenesis Kit was
purchased from Stratagene. Radiolabeled nucleotides were purchased from MP
Biomedicals. EnzChek Phosphate Assay Kit was purchased from Invitrogen. Glutathione
affinity column, Superdex 200 gel filtration column, PGEX-6P-1 vector and PreScission
protease were purchased from GE Healthcare.
74
Mutants Construction and Purification
LTag131-627 wild type and mutant were produced by E.Coli expression system
as described previously (Li et al., 2003). Briefly, the wild type or mutant protein was
expressed as a GST-LTag fusion using the PGEX-6P-1 vector, including a prescission or
cleavage site between GST and LTag. Fusion protein was purified through a glutathione
affinity column, and then GST was cleaved by PreScission protease. The LTag protein
was eluted from the affinity column then further purified by ion exchange and Superdex
200 gel filtration chromatography. The protein was concentrated to 5 mg/ml in a buffer
containing 25 mM Tris-Cl (pH8.0), 250 mM NaCl, and 10 mM DTT. All proteins were
quantified by using the Bradford method and Coomassie blue R-250 staining on SDS-
PAGE.
DNA Binding Assay
Rotational anisotropy was used to measure ssDNA and dsDNA binding by WT
and mutant LTag proteins. Measurements were taken on a QuantaMaster QM-1
fluorometer (Photon Technology International). Reaction mixtures (70 µl) contained 20
mM Tris (pH7.5), 10 mM MgCl
2
, 1 mM DTT, 0.1 mg/ml bovine serum albumin, 50 nM
DNA, LTag from 0 to 1,200 nM hexamer, and 1 mM adenosine 5’- (gamma-thio) tri-
phosphate (ATP-γ-S), if indicated, at 25°C. A 36-nucleotide ssDNA substrate labeled
with 5-carboxyfluorescein was used for all ssDNA anisotropy experiments. dsDNA
binding experiments used a 36-base-pair substrate labeled with 6-carboxyfluorescein at
the 5’ end of one strand; this substrate did not contain the viral origin. Nonlinear
75
regression was used to fit anisotropy data by the use of Enzfitter (Biosoft) to the
following equation (Greenleaf et al., 2008; Nakazawa et al., 2004):
r = (r
max
[E]
n
)/( K
d
n
+ [E]
n
) + r
m
(1)
where r, r
max
, and r
min
are the observed anisotropy, maximum anisotropy, and initial
anisotropy, respectively. [E] is the concentration of hexameric LTag, K
d
is the
concentration of LTag that produces 0.5r
max
, and n is the Hill coefficient.
ATPase Stimulation Assay
The ATPase assay was performed as described previously (Greenleaf et al.,
2008). The EnzChek phosphate assay kit was used according to the product manual
instruction. The ATPase assay was carried out in a reaction mixture containing 1mM
ATP and 0.7 µM (as monomer) of LTag WT or mutant in a 50 µl buffer (10 mM MgCl
2
,
20 mM Tris-Cl pH7.5), with 0.7 µM of ssDNA or dsDNA. After incubating for 10 min at
37°C, 50 µl of stop solution containing 20 mM Tris-Cl pH7.5, 200 mM EDTA, 0.4 mM
2-amino-6-mercapto-7-methylpurine riboside (MESG) and 2 U/ml Purine nucleoside
phosphorylase (PNPase) was added to the reaction and incubated for 30 min at 25°C. The
free phosphate released by ATP hydrolysis interacts with the MESG substrate and results
in a MESG spectrophotometric absorbance shift from 330 nm to 360 nm. The product
absorbance was read by spectrophotomer (Bio-Rad) at the wavelength of 360 nm. The
data from assay were quantified and plotted by Microsoft Excel.
76
Helicase Assay
For helicase assay, Y-shaped fork DNA with 44 nt single-stranded DNA tails and
a 44 nt duplex was obtained by annealing two oligonucleotides. The 146 bp double
stranded origin DNA substrate was prepared by PCR on the vector of PGEX-2TK with a
64 bp SV40 origin sequence insertion and the resulting sequence are shown in Table 3.2.
For the helicase assay, DNA substrate was 5’end-labeled with [γ-32P] ATP by T4
polynucleotide kinase. Approximately 10 to 15 fmol of labeled fork or origin DNA
substrate was incubated with 0.45 µM (as monomer for fork DNA) or 0.54 µM (as
monomer for origin DNA) of LTag WT or mutant in helicase buffer containing 20 mM
Tris-Cl pH7.5, 10 mM MgCl
2
, 5 mM ATP, 1 mM DTT and 0.1 mg/ml bovine serum
albumin for 45 min at 37°C. The reaction was terminated by adding to 0.1% SDS, 25 mM
EDTA and 10% glycerol. The sample was analyzed on 12% native polyacrylamide gel in
1X Tris-Borate-EDTA (TBE) buffer. The gel was dried and the radiolabeled
oligonucleotide was quantified by autoradiography. Data was quantified by Quantity One
and plotted by Microsoft Excel.
Biotin-Streptavidin Displacement Assay
Biotin-streptavidin displacement assay to detect origin DNA melting (or structure
distortion) was performed as described previously (Morris et al., 2002; Morris et al.,
2001). ssDNA and dsDNA substrates were synthesized with one biotin-dT in the middle.
The biotin substrate was 5′-radiolabeled with T4 polynucleotide kinase at 37°C for 30
min. Unincorporated [γ-32P] ATP was removed by passing the labeled oligonucleotides
77
through Bio-spin® P6 column. Approximately 10 to 15 fmol of labeled ss- or ds- DNA
substrate was incubated with 0.15 µM of Monovalent streptavidin in helicase buffer
containing 20 mM Tris-Cl pH7.5, 10 mM MgCl
2
, 5 mM ATP, 1 mM DTT and 0.1 mg/ml
bovine serum albumin for 5min at 37°C. Then add 0.09 - 0.36 µM LTag protein (WT or
mutant) with 0.9 µM of free biotin trap to the mixture to initiate the displacement assay.
The assay was performed at 25°C for 15 min and stopped by adding the quench solution
(1.5% SDS, 500 mM EDTA, pH 8.0, 0.2% xylene cyanol, 0.2% bromophenol blue, and
25% glycerol). Samples were analyzed by electrophoresis on a 12% polyacrylamide gel
in 1X TBE. The gel was dried and the radiolabeled oligonucleotide was quantified by
autoradiography.
Potassium Permanganate Reactivity Assay
Permanganate reactivity assay to test DNA melting activity was performed as
described previously (Liu et al., 2007; Sanders and Stenlund, 1998). A 100 bp origin
dsDNA was generated by PCR with one of the primers end-labeled with [γ-
32
P] ATP.
10~15 fmol DNA was incubated with 0.18 - 0.36 µM (as monomer) of LTag protein in
buffer containing 20 mM Tris-Cl pH7.5, 10m M MgCl
2
, 5 mM ADP (or AMP-PNP), 1
mM DTT and 0.1 mg/ml BSA for 15 min at 37°C. Then KMnO4 was added to a final
concentration of 6 mM and reactions was incubated at 37°C for 1 min. To stop the
oxidation reaction, a stop solution containing 80 mM β-mercaptoethanol, 0.3% SDS, and
10 mM EDTA. Modified DNA substrates were deproteinized by first digestion with
proteinase K (20 µg/ml, 60 min at 37°C) then binding to Qiagen QIAEX II beads.
78
Piperidine was added to 20% (v/v) on dried beads to cleave DNA substrates at modified
nucleotides (30 min at 90°C). The final product was run on 15% Urea-PAGE denaturing
gel for 45 min at 120 V. The gel was dried and the radiolabeled oligonucleotide was
quantified by autoradiography.
Table 3.2 DNA substrates used in assays
Table 3.2 DNA Substrates Used in Assays
a
: 64bp SV40 origin sequence is flanked by 41bp random DNA sequence
DNA%
NAMES%
DNA%SEQUENCES%
17nt
ssDNA
5’GAAGCCAATACAAAGGC
85bp
dsDNA
5’TCTTGCCCAACCCGTCTACACGCTGTTATAGCGAATCAGCGGGAACCCGGTGCCACGCGATGGAACGTCCTTAACTCTGGCAGGC
3’AGAACGGGTTGGGCAGATGTGCGACAATATCGCTTAGTCGCCCTTGGGCCACGGTGCGCTACCTTGCAGGAATTGAGACCGTCCG
Fork-
shaped
DNA
5’(dT)
44
GCTCGTGCAGACGTCGAGGTGAGGACGAGCTCCTCGTGACCACG
3’(dT)
44
CGAGCACGTCTGCAGCTCCACTCCTGCTCGAGGAGCACTGGTGC
146bp
Origin
DNA
a
5’(AC…AT)
41
CACTACTTCTGGAATAGCTCAGAGGCCGAGGCGGCCTCGGCCTCTGCATAAATAAAAAAAATTA(GT…GA)
41
3’(GT…AT)
41
GTGATGAAGACCTTATCGAGTCTCCGGCTCCGCCGGAGCCGGAGACGTATTTATTTTTTTTAAT(CA…CT)
41
ssDNA
with
Biotin
5’CAACGTATTCAAGATACCTCGTACTCTGTACBio-dTGACTGCGATCCGACTG
"
dsDNA
with
Biotin
5’CAACGTATTCAAGATACCTCGTACTCTGTACBio-dTGACTGCGATCCGACTG
3’GTTGCATAAGTTCTATGGAGCATGAGACATG A CTGACGCTAGGCTGAC"
100bp
origin
DNA
5’ACATGCACGAAACCAAGTCACTACTTCTGGAATAGCTCAGAGGCCGAGGCGGCCTCGGCCTCTGCATAAATAAAAAAAATTAGGCCGTATTAGTACAGTA
3’TGTACGTGCTTTGGTTCAGTGATGAAGACCTTATCGAGTCTCCGGCTCCGCCGGAGCCGGAGACGTATTTATTTTTTTTAATCCGGCATAATCATGTCAT"
79
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Abstract (if available)
Abstract
Simian Virus 40 (SV40) has been studied as the model system to elucidate the mechanism of eukaryotic DNA replication. The oncogenic large tumor antigen (LTag) encoded by SV40 not only transforms cells and induces tumors but also functions as a molecular motor machine that melts the viral origin and unwinds duplex DNA to initiate replication. It’s been regarded as the functional homologue of minichromosome maintenance (MCM) protein, a putative replicative helicase in eukaryotic and archaeal cells. ❧ Simian virus 40 large tumor antigen (LTag) is an AAA+ hexameric motor that harnesses the energy from ATP binding/hydrolysis to initiate DNA replication and unwind replication forks. However, how the six subunits of LTag hexamer motor coordinate for ATP hydrolysis and for DNA unwinding/translocation are unresolved. Here I investigated the subunit coordination mechanisms for ATP hydrolysis and DNA unwinding through a series of mutant doping experiments. For ATP hydrolysis, I observed a random mode in the absence of DNA, a semi-random mode with ssDNA, and a semi-coordinated mode with fork or origin DNA. For DNA unwinding, however, the results indicated a semi-coordinated mode for fork-DNA, but a fully coordinated mode for origin DNA. These results and previous evidence suggest a distinctive coordination behavior for LTag, which adopts different coordination for ssDNA translocation, fork- DNA unwinding, and origin DNA unwinding. For origin DNA unwinding, LTag hexamer operates in a fully coordinated mode. ❧ The β-hairpin and F-loop in the central channel of LTag hexamer has been identified to play a key role in interacting with DNA substrate. Here, I made a series of mutations on the tip of β-hairpin and an adjacent F-loop structure. A variety of functional assays have been performed using these mutants. I demonstrate that two neighboring β- hairpin tip residues (K512 and H513) and residue F459 on F-loop are involved in DNA- dependent ATPase stimulation, in local origin melting, ss- and ds- DNA translocation, and fork- and origin- DNA unwinding. However, their relative functional roles in these activities are different, with K512 as a sensor to recognize and convey the DNA signal from the central channel while H513 and F459 as mechanics to interact with DNA base pairs for melting and unwinding. The biochemical results support the structural observations that the positively charged residue K512 interacts with DNA phosphate backbone through electrostatic interactions while ring-shaped residues H513 and F459 interact with DNA by hydrophobic stacking. ❧ In summary, my thesis is focused on the functional study of LTag in regard to ATP hydrolysis and DNA unwinding, as well as the role defining of important residues on β-hairpin and loop structure. The overall work will provide important clues towards studying other motor helicases in the same family and insights into the big picture of DNA initiation and replication.
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Yu, Xian Jessica
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Mechanism study of SV40 large tumor antigen atpase and helicase functions in viral DNA replication
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College of Letters, Arts and Sciences
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Doctor of Philosophy
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Molecular Biology
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hexameric helicase,large T antigen,OAI-PMH Harvest,subunit coordination mechanism,SV40 replication
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hexameric helicase
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subunit coordination mechanism
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