Virology 384 (2009) 352–359
Contents lists available at ScienceDirect
Virology
j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / y v i r o
Minireview
SV40 DNA replication: From the A gene to a nanomachine
Ellen Fanning ⁎, Kun Zhao
Department of Biological Sciences and Vanderbilt-Ingram Cancer Center, Vanderbilt University, VU Station B 351634, Nashville, TN 37235-1634, USA
a r t i c l e
i n f o
Article history:
Received 11 November 2008
Accepted 18 November 2008
Available online 20 December 2008
Keywords:
Genome structure
DNA replication origin
T antigen
Helicase
Double hexamer
Replication fork
Hand-off
Polymerase switch
DNA damage signaling
a b s t r a c t
Duplication of the simian virus 40 (SV40) genome is the best understood eukaryotic DNA replication process
to date. Like most prokaryotic genomes, the SV40 genome is a circular duplex DNA organized in a single
replicon. This small viral genome, its association with host histones in nucleosomes, and its dependence on
the host cell milieu for replication factors and precursors led to its adoption as a simple and powerful model.
The steps in replication, the viral initiator, the host proteins, and their mechanisms of action were initially
defined using a cell-free SV40 replication reaction. Although our understanding of the vastly more complex
host replication fork is advancing, no eukaryotic replisome has yet been reconstituted and the SV40 paradigm
remains a point of reference. This article reviews some of the milestones in the development of this paradigm
and speculates on its potential utility to address unsolved questions in eukaryotic genome maintenance.
© 2008 Elsevier Inc. All rights reserved.
Introduction
The study of bacteriophage and viruses over the past 50 years laid
the foundations of modern molecular biology. The physicists,
chemists, biologists, and physicians pioneered this frontier with the
hope that the relative simplicity of these agents might allow them to
serve as tools to understand their vastly more complex infected host
cells. The discovery of simple DNA viruses that propagated in
mammalian cell nuclei and caused tumors in experimental animals
led the way to an explosion of eukaryotic molecular biology and its
applications to understanding, treating, and preventing human
disease. For pioneering studies of the DNA tumor viruses polyomavirus
and SV40 in the 1969's, Renato Dulbecco was awarded the 1975 Nobel
Prize in Physiology or Medicine. Equally importantly, the many young
scientists who trained in his laboratory were inspired to pursue and
expand on this fruitful approach in ever more exciting new directions.
The development of the field and collegial interactions among
members of the DNA tumor virus community were greatly fostered
by annual meetings sponsored by Cold Spring Harbor Laboratory and
Imperial Cancer Research Fund, as well as by review volumes edited by
John Tooze beginning in 1973 (Tooze, 1973).
This article reviews some of the fundamental lessons on genome
structure, DNA replication, and genome maintenance that these
deceptively simple viruses have revealed over the past 4 decades.
The utility of these viral paradigms in guiding the investigation of
mammalian DNA replication is considered. The article concludes with
⁎ Corresponding author. Fax: +1 615 343 6707.
E-mail address: [email protected] (E. Fanning).
0042-6822/$ – see front matter © 2008 Elsevier Inc. All rights reserved.
doi:10.1016/j.virol.2008.11.038
reflections on how the rapidly growing understanding of host genome
maintenance is leading to a re-consideration of how these viruses
exploit their host cells.
The SV40 minichromosome: genetic and physical maps linked
through DNA sequence
The SV40 genome is a covalently closed circular duplex DNA
molecule of 3.6 × 106 Da (Crawford and Black, 1964; Dulbecco and Vogt,
1963; Weil and Vinograd, 1963). Biophysical characterization of
superhelical SV40 and polyomavirus DNA provided the first insight
into the initially puzzling ability of supercoiled DNA to renature rapidly
after exposure to alkali (Vinograd et al., 1965; Weil, 1963; Weil and
Vinograd, 1963), its limited uptake of intercalating dyes, e.g. ethidium
bromide, and other properties typical of supercoiled DNA. The SV40
genome exists in the virus particle and in infected cells as a minichromosome packaged with host cell histones into nucleosomes that
closely resemble those of the host chromatin (Bonner et al., 1968;
Germond et al., 1975; Griffith, 1975; White and Eason, 1971) (Fig. 1).
Purification of SV40 DNA from minichromosomes reveals its negatively
supercoiled topology. As we now know, this topology endows
chromatin with the capacity to readily denature for initiation of DNA
replication or transcription.
Genetic studies of replication in prokaryotes had led to a
potentially general model for control of replication: the replicon
model of Jacob, Brenner, and Cuzin (Jacob and Brenner, 1963). The
model postulated a cis-acting element, the replicator, recognized by a
trans-acting factor, the initiator. This interaction would lead to locally
denatured duplex DNA in or near the replicator element and initiation
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Fig. 1. Electron micrograph of an SV40 minichromosome isolated from productively
infected cells. (Scale bar 100 nm). (Reprinted from Griffith, 1975 with permission
from AAAS.)
of replication. Each replicator with its initiator would thus govern the
replication of the flanking regions of DNA, the replicon. If this model
were general, one might expect eukaryotic DNA to be organized into
replicons in a similar manner. If SV40 DNA represents a eukaryotic
replicon, one would predict a genetically definable viral replicator
element and an initiator that recognized it.
In the mind of Daniel Nathans at Johns Hopkins University, the
appeal of SV40 as an object for genetic analysis converged with the
discovery of the first sequence-specific restriction endonucleases by
his colleague Hamilton Smith (Fig. 2). Nathans and colleagues
generated the first restriction cleavage map of SV40 DNA, by
determining the physical order of the Hind II/III and Hpa I/II sites
around the SV40 DNA genome (Danna and Nathans, 1971; Danna et al.,
1973). A unique restriction cleavage site by Eco RI (Morrow and Berg,
1972) provided a point of reference in the viral genome. By 1972,
Danna and Nathans had combined a radiolabeled thymidine pulsechase approach with their restriction map to determine the physical
start site for SV40 DNA replication, the origin, and show that
replication proceeded bidirectionally to terminate on the opposite
side of the DNA molecule (Danna and Nathans, 1972; Nathans and
Danna, 1972). [For a fascinating overview of these discoveries, see
Brownlee, 2005; Roberts, 2005] This physical map greatly facilitated
determination of the 5243 bp sequence of SV40 DNA, the first
eukaryotic genome to be completely sequenced (Fiers et al., 1978;
Reddy et al., 1978). Moreover, the map and the sequence enabled the
classical mutational analysis of the SV40 genome (Chou and Martin,
1974; Tegtmeyer, 1972; Tegtmeyer and Ozer, 1971) to be correlated
with nucleotide sequence changes that affected viral DNA replication
(temperature-sensitive complementation group A (tsA)), cell transformation (tsA), and virion production (tsB, C, BC, D) [for a personal
account, see Nathans, 1978].
With the viral DNA sequence in hand and new restriction
endonucleases rapidly emerging in several laboratories, the Nathans
lab moved quickly to test the function of the SV40 origin of DNA
replication by mutational analysis. They devised site-directed
mutagenesis protocols for deletions and base substitutions followed
by selection for resistance to cleavage by Bgl I, which has a single
recognition site in SV40 DNA at the origin (DiMaio and Nathans, 1980;
DiMaio and Nathans, 1982; Shortle and Nathans, 1978). These
mutations were mapped by DNA sequencing and shown to render
SV40 replication defective when the genome was introduced into host
cells, satisfying one criterion for a replicator element. To examine the
relationship between the putative replicator element and the tsA gene
that was also involved in replication, the Nathans lab carried out a
mutational screen for second site revertants of the replication-
353
defective mutant origins. These pseudorevertant mutations were
then mapped and shown to reside at positions outside of the origin
region and to alter the coding sequence of the tsA gene (Margolskee
and Nathans, 1984; Shortle et al., 1979). The A gene encodes the SV40
large tumor (T) antigen (Tag), a multifunctional protein whose
structure and roles in viral DNA replication are reviewed below.
Thus, the origin element interacted genetically with a viral gene that
regulated the rate of viral DNA replication, providing strong evidence
for a replicon model in controlling replication of SV40 DNA.
Biochemical investigation of Tag promptly confirmed the interaction,
paving the way for new experiments to elucidate the mechanism of
SV40 DNA replication.
Further dissection of the viral replicator in multiple laboratories
revealed a 64 bp core composed of three elements. A central element
contains a palindromic array of four GAGGC pentanucleotides that, as
we now know, serve as binding sites for Tag. The binding sites are
flanked by an easily denatured imperfect palindrome (EP) on one side
and by an AT-rich sequence on the other side. The two flanking
elements undergo local distortion or melting during initiation of
replication (Borowiec et al., 1990). The early and late promoter
elements flank the viral core origin and stimulate its activity in
infected cells, as does the viral enhancer element. These auxiliary
elements may stimulate initiation of replication from the viral core
origin at multiple levels, some of which may reflect the close
relationship between origins of replication and transcription. The
first level may be by modulating the structure of the core origin DNA
to facilitate distortion by Tag, e.g. through intrinsically bent AT-rich
DNA sequences, or local modulation of supercoiling. These physical
properties of origin DNA are also found in eukaryotic chromosomal
origins of replication and are thought to be important for binding of
the origin recognition complex ORC (Remus et al., 2004). Proteins
bound to the auxiliary elements may also facilitate Tag assembly on
the core origin DNA, Tag remodeling into an active helicase, or
recruitment of host replication proteins. For example, chromatin
remodeling to generate a nucleosome-free origin region and histone
modifications are likely to be important for initiation at the SV40
origin and at chromosomal origins (Saragosti et al., 1980). Lastly,
replication of the viral minichromosome appears to take place in
specific subnuclear domains (Ishov and Maul, 1996; Staufenbiel and
Deppert, 1983; Tang et al., 2000), but how the minichromosome is
targeted to these sites remains poorly defined.
Given the sequence specificity of the SV40 core origin and the
dependence of the virus on host cell proteins for DNA replication, it
was tempting to imagine that chromosomal origins of replication
might also be composed of modules with defined sequences that
Fig. 2. Daniel Nathans (left) and Hamilton Smith in the laboratory at Johns Hopkins
University. (Reprinted from Roberts, 2005 with permission from Copyright 2005
National Academy of Sciences, U.S.A.)
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could be recognized by initiator proteins. Although origins in budding
yeast fulfill this expectation to some extent, no common consensus
sequence has been found in either the genetically defined replicators
or the start sites of replication in higher eukaryotic genomes (Aladjem,
2007; Aladjem and Fanning, 2004; Bell, 2002).
SV40 large T antigen (Tag): the initiator protein in viral DNA
replication
Tag was first detected as a 90–100 kDa polypeptide from infected
cell extracts that reacted with the serum of rodents bearing tumors
induced by SV40 injection (Rundell et al., 1977). Initially it was
uncertain whether Tag was in fact encoded by the A gene in the SV40
genome since small deletions in that gene failed to reduce the
apparent mass of the immunoreactive protein. This conundrum was
resolved through the combined efforts of the Tegtmeyer, Crawford and
Berg labs (Fig. 3) (Crawford et al., 1978), providing the first hint that
Tag was expressed from one of the three alternatively spliced early
SV40 transcripts (see Yaniv article in this issue).
Taking advantage of the high level expression of a Tag-related
adenovirus-SV40 hybrid protein D2, Robert Tjian succeeded in a
classical purification of the first native, biochemically active form of
Tag (Tjian, 1978). This key achievement was the first step toward
defining the mechanism of SV40 DNA replication. Tjian showed that
purified D2 bound to DNA and was capable of specifically protecting
SV40 origin DNA sequences against nuclease digestion, confirming the
genetic interactions between the SV40 origin and the A gene encoding
Tag. Moreover, D2 bound sequentially to several elements of the viral
origin DNA in a manner suggesting possible multimerization of the
protein on the DNA to protect up to 120 bp. Subsequent work in
multiple laboratories led to definition of the pentanucleotide GAGGC
as the fundamental recognition motif specifically bound by Tag, either
as a tandem repeat separated by 7 bp of intrinsically bent DNA (site I)
or, in the core origin, as a 27 bp palindromic arrangement of 4
pentanucleotides separated by 1 bp (site II) (reviewed by Borowiec
et al., 1990; Challberg and Kelly, 1989; Fanning and Knippers, 1992;
Stillman, 1989).
Purified D2 and Tag were soon shown to display a second
biochemical activity: the ability to bind and hydrolyze Mg-ATP/dATP,
an activity stimulated by single-stranded DNA (Cole et al., 1986;
Giacherio and Hager, 1979). Although this behavior suggested that Tag
might have DNA helicase activity, it could not be convincingly
demonstrated. Using Tag purified from infected cells by immunoaffinity
chromatography on monoclonal antibody resins (Deppert et al., 1981;
Dixon and Nathans, 1985; Harlow et al., 1981; Simanis and Lane, 1985)
and a clever new helicase assay (Hubscher and Stalder, 1985), the
Knippers lab showed that highly purified Tag could unwind partial
Fig. 3. Peter Tegtmeyer (left) in discussion at a DNA Tumor Virus meeting, University of
Wisconsin-Madison (Courtesy of K. Rundell).
Fig. 4. Rolf Knippers in his laboratory at the University of Konstanz, Germany, in the
1980's. (Courtesy of M. Baack).
duplex DNA with 3′ to 5′ polarity in an ATP-hydrolysis dependent
manner (Fig. 4) (Stahl et al., 1986). Moreover, monoclonal antibodies
against specific regions of Tag inhibited helicase activity and mutations
in Tag that reduced ATPase activity also reduced helicase activity (Stahl
et al.,1986). The helicase activity of Tag was quickly confirmed in several
other laboratories (reviewed in Fanning and Knippers, 1992).
Despite this important step in understanding the role of Tag in viral
DNA replication, it remained unclear how the helicase activity of Tag
could unwind duplex DNA from the origin. The Hurwitz laboratory,
collaborating with several others, discovered that Tag assembles into a
multimer on the viral origin in an ATP-binding dependent manner to
form a bilobed double hexameric structure that distorts the duplex
DNA locally (Borowiec et al., 1990; Dodson et al., 1987; Mastrangelo
et al., 1989). In the presence of Mg-ATP, a single-stranded DNA binding
protein, and topoisomerase I, Tag generated a theta-like structure with
single-stranded bubbles of varying sizes with the origin always at the
center of the bubble (Dean et al., 1987a; Dean et al., 1987b; Dodson
et al., 1987). A large protein complex likely to be Tag hexamer was
often observed at the junctions of the single-stranded bubble with
duplex DNA, suggesting that two diverging Tag hexamers unwound
the duplex at a similar rate. A few years later, under different
experimental conditions, active unwinding complexes of double
hexamer were visualized with two loops of ssDNA emanating from
the double hexamer (Fig. 5). These images suggested a more
sophisticated bidirectional unwinding mechanism in which duplex
parental DNA is reeled into the double hexamer, coordinately from
both sides, and the unwound template is spooled out for replication
(Wessel et al., 1992). This type of unwinding intermediate, not
observed in initiation of prokaryotic replication by 5′ to 3′ helicases
(Fang et al., 1999), implied some kind of functional contacts between
Tag hexamers. Biochemical, genetic, and structural data (Meinke et al.,
2006; Moarefi et al., 1993; Smelkova and Borowiec, 1998; Valle et al.,
2006; Valle et al., 2000; Virshup et al., 1992; Weisshart et al., 1999)
provide evidence for functional interactions of Tag residues 102–259
in one hexamer with the corresponding residues in the other
hexamer, strongly supporting this model of bidirectional unwinding
as physiologically relevant (reviewed in Bullock, 1997; Fanning, 1994;
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355
Fig. 7. A minimal set of replication proteins at a eukaryotic fork. MCM helicase substitutes
here for Tag; the PCNA clamp loader RFC and topoisomerases are not shown. (Reprinted
from Garg and Burgers, 2005 with permission from Copyright 2005 Taylor and Francis.)
Fig. 5. Active unwinding from the SV40 origin by a Tag double hexamer. Tag was
incubated under unwinding conditions with a duplex plasmid DNA fragment containing the SV40 origin 1.1 or 1.5 kb from the ends. The reaction was terminated by
glutaraldehyde, the sample was purified, spread, negatively stained, and visualized by
electron microscopy. Two single-stranded loops bound to single-stranded DNA binding
protein emanate from a Tag double hexamer (see inset). (Scale bar 100 nm). The double
hexamer and the loops dissociated into a theta-like structure after treatment with EDTA
(not shown). (Reprinted from Wessel et al., 1992 with permission from Copyright 1992
American Society for Microbiology.)
Simmons, 2000). This model suggests that both replication forks may
assemble, at least initially, on the Tag double hexamer and that
progression of the two forks may be coupled (Falaschi, 2000).
However, this possibility has not been addressed experimentally.
In parallel with studies of Tag helicase activity, a trio of laboratories
embarked on a major quest to establish a cell-free SV40 DNA system
that could be used to identify the host proteins necessary to replicate
viral DNA. Early work with replicating SV40 nucleoprotein complexes
isolated from infected cells had already been shown to complete
replication in cell-free extracts (DePamphilis et al., 1975; Edenberg
et al., 1976) and DNA polymerase alpha-primase had been identified as
a key activity (Otto and Fanning, 1978; Waqar et al., 1978). Joachim Li
and Thomas Kelly (1984)were the first to succeed in using purified
SV40 origin DNA and extracts from primate cells supplemented with
immunopurified Tag to replicate a DNA template from the viral origin,
followed quickly by independent studies in the Stillman and Hurwitz
laboratories (Li and Kelly, 1984; Stillman and Gluzman, 1985; Wobbe
et al., 1985) (Figs. 6, 7). Countless hours of painstaking work in cold
rooms in these and other labs resulted in the purification of ten human
proteins that, together with Tag, are sufficient to reconstitute the
replication of duplex plasmid DNA in an origin-dependent reaction
(Waga and Stillman, 1994; Waga and Stillman, 1998).
To elucidate the basic mechanisms of replication, sub-reactions for
individual steps were reconstituted with purified proteins. Three
stages of the replication process have been reconstituted and studied
in detail: initiation, elongation, and Okazaki fragment maturation.
Four proteins (Tag, RPA, DNA polymerase alpha-primase, and
topoisomerase I) are sufficient to reconstitute initiation of replication
at the viral origin (Matsumoto et al., 1990) reviewed by Borowiec et al.,
(1990) and Bullock, (1997). After primer synthesis, the clamp-loader
replication factor C (RFC) orchestrates a switch from DNA polymerase
alpha-primase to the more processive DNA polymerase delta
(Tsurimoto et al., 1990; Tsurimoto and Stillman, 1991; Weinberg
et al., 1990) (Fig. 8). RPA contacts with RFC appear to play a role in
displacing DNA polymerase alpha-primase from the template
(Yuzhakov et al., 1999). RFC bound to ATP binds the sliding clamp
PCNA, cracks open the ring, and RFC contact with primer-template
triggers ATP hydrolysis, releasing a closed PCNA complex loaded at the
primer-terminus and able to bind and position polymerase delta for
primer extension (Indiani and O'Donnell, 2006).
Despite the simplicity of the SV40 replication fork and several key
differences relative to host replication forks and their intricate
regulatory wiring, the SV40 fork continues to serve as a useful
paradigm for host forks (Figs. 7, 8), where new challenges await.
Structures of many of these host replication proteins and their
domains have now been determined by X-ray crystallography (for
examples, see Bowman et al., 2004; Fanning et al., 2006; Garg and
Burgers, 2005; Indiani and O'Donnell, 2006; Pascal et al., 2004),
opening the possibility of developing an atomic level understanding of
replication fork operation in eukaryotes. Definition of how these
exchanges of proteins and coupling of leading and lagging strand
replication are accomplished will require more complete structural
information on DNA polymerases, definition of protein interactions
required for hand-off reactions, and single molecule studies.
Seeing is believing: structures of SV40 Tag
Fig. 6. Thomas J. Kelly (left) and Bruce Stillman (right) at a reception during the 1994
Cold Spring Harbor Symposium (Courtesy of Cold Spring Harbor Archives).
Mapping of functional domains of Tag by molecular genetics and
biochemistry provided the first hints that SV40 Tag is a multi-domain
356
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Fig. 10. Crystallographer Xiaojiang S. Chen, University of Southern California.
Fig. 8. Linking the polymerase switch to Okazaki fragment processing. The mechanism
of elongation of a primed DNA template on the leading strand or for each Okazaki
fragment on the lagging strand was studied in reactions reconstituted with purified
proteins (steps 1–4). The mechanism of Okazaki fragment maturation was also
elucidated in reconstituted reactions (steps 5–7). (Reprinted from Waga and Stillman,
1998 with permission from Annual Reviews.) (For a current view of Okazaki fragment
processing, see Rossi et al., 2008).
protein with a bewildering number of functions (Fanning, 1992).
Mapping of structured domains by limited proteolysis correlated to
some extent with the functional domain map (Schwyzer et al., 1980),
but an atomic structure of Tag remained elusive until the focus shifted
to the protein domains. At last, the origin DNA binding domain (OBD)
was determined using NMR in the Bullock and Bachovchin laboratories
(Fig. 9) (Luo et al., 1996). These structural studies, together with
previously mapped mutations that affected origin DNA binding,
provided important insight into the mechanism of origin recognition
by Tag and assembly of the double hexamer (Bullock, 1997; Joo et al.,
1998). The next domain to come into view was the DnaJ co-chaperone
domain (Fig. 9) in complex with a fragment of the retinoblastoma
tumor suppressor protein (Kim et al., 2001; reviewed by Hennessy et al.,
2005; Sullivan and Pipas, 2002). Although the DnaJ function is not
Fig. 9. 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. (Courtesy of X.S. Chen.)
required for SV40 replication in cell-free reactions (Weisshart et al.,
1996), its functions are essential for DNA replication in productively
infected cells (Campbell et al., 1997). In a key advance for understanding
the mechanism of initiation of viral DNA replication, the first crystal
structure of the Tag helicase domain was determined in Xiaojiang
Chen's laboratory (Fig. 10) (Li et al., 2003). The structured domain
(residues 251–627) consists of the zinc-binding and ATPase/AAA+
subdomains, comprising about two-thirds of the protein (Fig. 9). The
origin DNA binding domain and the DnaJ domain are tethered to one
another and to the helicase domain through flexible linkers (Fig. 9).
The structure of the host range domain (residues 628–708) remains
unknown (see article by Pipas in this issue). These structured domains
correlate very well with the functional domains of Tag deduced by
biochemistry and molecular genetics (see Fanning and Knippers,
1992; Weisshart et al., 2004). Remarkably, the Tag helicase structure is
closely related to that of the chromosomal replicative helicase, as
evidenced by crystal structures of archaeal MCM helicase domains
(Fletcher et al., 2003; Gomez-Llorente et al., 2005; reviewed by
Sclafani and Holzen, 2007).
The Tag structures available so far suggest a modular molecule that
likely operates dynamically to coordinate origin DNA binding and
local distortion by the double hexamer, and re-organization of the
double hexamer into a bidirectional unwinding machine (Bochkareva
et al., 2006; Gai et al., 2004; Meinke et al., 2006; Meinke et al., 2007;
Valle et al., 2006). Re-examination of the initiation reaction and
mapping of protein–protein interactions at the atomic level is
beginning to provide insight into the dynamic nature of the initiation
process. During initiation of replication, RPA association with Tag-OBD
couples origin DNA unwinding to loading of RPA on the emerging
template (Jiang et al., 2006). Similarly, Tag-OBD-mediated remodeling
of RPA-ssDNA complexes facilitates the ssDNA hand-off to the DNA
polymerase alpha-primase positioned on the Tag helicase domain
(Arunkumar et al., 2005). Nevertheless, until the relative positions of
the Tag domains in the double hexamer and the path of the DNA
template through the protein during various functional states of
replication can be visualized, our understanding of the SV40
replication process is incomplete. One successful path toward this
ultimate goal began by analyzing electron micrographs of Tag double
hexamers on origin DNA with image processing algorithms (Valle
et al., 2000). Recent advances in cryo-electron microscopy of wild type
and mutant Tag on origin DNA, new image classification algorithms,
and fitting of new atomic structures of Tag may allow this goal to be
reached.
The SV40 replication paradigm: a perspective
Much effort has been aimed at elucidating in detail the operation
of the SV40 replisome with the hope that general principles can be
Minireview
discerned that will increase our understanding of mammalian
chromosomal replication. The bidirectional “replication factory”
organization of the SV40 replisome appears to be a simple example
of those that duplicate bacterial and eukaryotic chromosomes
(Kitamura et al., 2006; Meister et al., 2006). However, SV40 and host
DNA replication mechanisms differ in several major features. SV40
encodes its own DNA helicase, whereas chromosomal replication
depends on the Cdc45/Mcm2-7/GINS helicase (Moyer et al., 2006 and
references therein). DNA polymerase epsilon is important for
chromosomal replication (Seki et al., 2006; Shikata et al., 2006), but
SV40 replication does not utilize it in vivo or in vitro (Pospiech et al.,
1999; Zlotkin et al., 1996). SV40 DNA replicates during the S/G2 phase
of the cell cycle, but DNA polymerase alpha-primase phosphorylated
by cyclin-dependent kinase is unable to support replication of viral
DNA in a cell-free reaction (Ott et al., 2002 and references therein).
Importantly, SV40 and polyomavirus infection induce DNA damage
signaling that promotes viral DNA replication (Dahl et al., 2005; Shi
et al., 2005; Wu et al., 2004; Zhao et al., 2008), but ordinarily inhibits
chromosomal replication and cell cycle transitions (Cimprich and
Cortez, 2008; Lavin, 2008). Because of these differences, one of the
most critical open questions is whether the SV40 replisome operates
as a streamlined mimic of host chromosomal replication or rather, a
host DNA repair pathway. The unanticipated role of DNA damage
signaling in viral DNA replication raises the question of whether other
undiscovered factors and mechanisms might participate in SV40
replication in infected cells, including the Hsc70 interaction with the
Tag DnaJ domain, sister cohesion and chromosome segregation,
subnuclear positioning of viral genomes, interaction with the
ubiquitin-proteasome system, and chromatin modification. The
SV40 model, with its rich history and wealth of experimental tools,
may have an exciting future.
Acknowledgments
EF thanks her mentors, lab members, colleagues, collaborators, and
fellow travelers along the SV40 road for fruitful cooperation, companionship, guidance, and inspiration. B. Stillman, C. Clark, K. Rundell, X.S.
Chen, and M. Baack kindly provided photographs. Financial support
from NIH (GM52948 to EF and P30 CA68485 to the Vanderbilt-Ingram
Cancer Center) and Vanderbilt University are gratefully acknowledged.
References
Aladjem, M.I., 2007. Replication in context: dynamic regulation of DNA replication
patterns in metazoans. Nat. Rev. Genet. 8 (8), 588–600.
Aladjem, M.I., Fanning, E., 2004. The replicon revisited: an old model learns new tricks
in metazoan chromosomes. EMBO Rep. 5 (7), 686–691.
Arunkumar, A.I., Klimovich, V., Jiang, X., Ott, R.D., Mizoue, L., Fanning, E., Chazin, W.J.,
2005. Insights into hRPA32 C-terminal domain-mediated assembly of the simian
virus 40 replisome. Nat. Struct. Mol. Biol. 12 (4), 332–339.
Bell, S.P., 2002. The origin recognition complex: from simple origins to complex
functions. Genes Dev. 16 (6), 659–672.
Bochkareva, E., Martynowski, D., Seitova, A., Bochkarev, A., 2006. Structure of the originbinding domain of simian virus 40 large T antigen bound to DNA. EMBO J. 25 (24),
5961–5969.
Bonner, J., Dhamus, M.E., Fambrough, D., Huang, R.C., Marushige, K., Tuan, D.Y., 1968. The
biology of isolated chromatin. Chromosomes, biologically active in the test tube,
provide a powerful tool for the study of gene action. Science 159 (810), 47–56.
Borowiec, J.A., Dean, F.B., Bullock, P.A., Hurwitz, J., 1990. Binding and unwinding—how T
antigen engages the SV40 origin of DNA replication. Cell 60 (2), 181–184.
Bowman, G.D., O, Donnell, M., Kuriyan, J., 2004. Structural analysis of a eukaryotic
sliding DNA clamp-clamp loader complex. Nature 429 (6993), 724–730.
Brownlee, C., 2005. Danna and Nathans: restriction enzymes and the boon to modern
molecular biology. Proc. Natl. Acad. Sci. U. S. A. 102 (17), 5909.
Bullock, P.A., 1997. The initiation of simian virus 40 DNA replication in vitro. Crit. Rev.
Biochem. Mol. Biol. 32 (6), 503–568.
Campbell, K.S., Mullane, K.P., Aksoy, I.A., Stubdal, H., Zalvide, J., Pipas, J.M., Silver, P.A.,
Roberts, T.M., Schaffhausen, B.S., DeCaprio, J.A., 1997. DnaJ/hsp40 chaperone
domain of SV40 large T antigen promotes efficient viral DNA replication. Genes
Dev. 11 (9), 1098–1110.
Challberg, M.D., Kelly, T.J., 1989. Animal virus DNA replication. Annu. Rev. Biochem. 58,
671–717.
357
Chou, J.Y., Martin, R.G.,1974. Complementation analysis of simian virus 40 mutants. J. Virol.
13 (5), 1101–1109.
Cimprich, K.A., Cortez, D., 2008. ATR: an essential regulator of genome integrity. Nat.
Rev. Mol. Cell Biol. 9 (8), 616–627.
Cole, C.N., Tornow, J., Clark, R., Tjian, R., 1986. Properties of the simian virus 40 (SV40)
large T antigens encoded by SV40 mutants with deletions in gene A. J. Virol. 57 (2),
539–546.
Crawford, L.V., Black, P.H., 1964. The nucleic acid of simian virus 40. Virology 24, 388–392.
Crawford, L.V., Cole, C.N., Smith, A.E., Paucha, E., Tegtmeyer, P., Rundell, K., Berg, P., 1978.
Organization and expression of early genes of simian virus 40. Proc. Natl. Acad. Sci.
U. S. A. 75 (1), 117–121.
Dahl, J., You, J., Benjamin, T.L., 2005. Induction and utilization of an ATM signaling
pathway by polyomavirus. J. Virol. 79 (20), 13007–13017.
Danna, K., Nathans, D., 1971. Specific cleavage of simian virus 40 DNA by restriction
endonuclease of Hemophilus influenzae. Proc. Natl. Acad. Sci. U. S. A. 68 (12),
2913–2917.
Danna, K.J., Nathans, D., 1972. Bidirectional replication of simian virus 40 DNA. Proc.
Natl. Acad. Sci. U. S. A. 69 (11), 3097–3100.
Danna, K.J., Sack Jr., G.H., Nathans, D., 1973. Studies of simian virus 40 DNA. VII. A
cleavage map of the SV40 genome. J. Mol. Biol. 78 (2), 363–376.
Dean, F.B., Borowiec, J.A., Ishimi, Y., Deb, S., Tegtmeyer, P., Hurwitz, J., 1987a. Simian virus
40 large tumor antigen requires three core replication origin domains for DNA
unwinding and replication in vitro. Proc. Natl. Acad. Sci. U. S. A. 84 (23), 8267–8271.
Dean, F.B., Bullock, P., Murakami, Y., Wobbe, C.R., Weissbach, L., Hurwitz, J., 1987b.
Simian virus 40 (SV40) DNA replication: SV40 large T antigen unwinds DNA
containing the SV40 origin of replication. Proc. Natl. Acad. Sci. U. S. A. 84 (1), 16–20.
DePamphilis, M.L., Beard, P., Berg, P., 1975. Synthesis of superhelical simian virus 40
deoxyribonucleic acid in cell lysates⁎. J. Biol. Chem. 250 (11), 4340–4347.
Deppert, W., Gurney, E.G., Harrison, R.O., 1981. Monoclonal antibodies against simian
virus 40 tumor antigens: analysis of antigenic binding sites, using adenovirus type
2-simian virus 40 hybrid viruses. J. Virol. 37 (1), 478–482.
DiMaio, D., Nathans, D., 1980. Cold-sensitive regulatory mutants of simian virus 40. J. Mol.
Biol. 140 (1), 129–142.
DiMaio, D., Nathans, D., 1982. Regulatory mutants of simian virus 40. Effect of mutations
at a T antigen binding site on DNA replication and expression of viral genes. J. Mol.
Biol. 156 (3), 531–548.
Dixon, R.A., Nathans, D., 1985. Purification of simian virus 40 large T antigen by
immunoaffinity chromatography. J. Virol. 53 (3), 1001–1004.
Dodson, M., Dean, F.B., Bullock, P., Echols, H., Hurwitz, J., 1987. Unwinding of duplex DNA
from the SV40 origin of replication by T antigen. Science 238 (4829), 964–967.
Dulbecco, R., Vogt, M., 1963. Evidence for a ring structure of polyoma virus DNA. Proc.
Natl. Acad. Sci. U. S. A. 50, 236–243.
Edenberg, H.J., Waqar, M.A., Huberman, J.A., 1976. Subnuclear systems for synthesis of
simian virus 40 DNA in vitro. Proc. Natl. Acad. Sci. U. S. A. 73 (12), 4392–4396.
Falaschi, A., 2000. Eukaryotic DNA replication: a model for a fixed double replisome.
Trends Genet. 16 (2), 88–92.
Fang, L., Davey, M.J., O, Donnell, M., 1999. Replisome assembly at oriC, the replication
origin of E. coli, reveals an explanation for initiation sites outside an origin. Mol. Cell
4 (4), 541–553.
Fanning, E., 1992. Simian virus 40 large T antigen: the puzzle, the pieces, and the
emerging picture. J. Virol. 66 (3), 1289–1293.
Fanning, E., 1994. Control of SV40 DNA replication by protein phosphorylation: a model
for cellular DNA replication? Trends Cell Biol. 4 (7), 250–255.
Fanning, E., Knippers, R., 1992. Structure and function of simian virus 40 large tumor
antigen. Annu. Rev. Biochem. 61, 55–85.
Fanning, E., Klimovich, V., Nager, A.R., 2006. A dynamic model for replication protein A
(RPA) function in DNA processing pathways. Nucleic Acids Res. 34 (15), 4126–4137.
Fiers, W., Contreras, R., Haegemann, G., Rogiers, R., Van de Voorde, A., Van Heuverswyn, H.,
Van Herreweghe, J., Volckaert, G., Ysebaert, M., 1978. Complete nucleotide sequence
of SV40 DNA. Nature 273 (5658), 113–120.
Fletcher, R.J., Bishop, B.E., Leon, R.P., Sclafani, R.A., Ogata, C.M., Chen, X.S., 2003. The
structure and function of MCM from archaeal M. Thermoautotrophicum. Nat. Struct.
Biol. 10 (3), 160–167.
Gai, D., Zhao, R., Li, D., Finkielstein, C.V., Chen, X.S., 2004. Mechanisms of conformational
change for a replicative hexameric helicase of SV40 large tumor antigen. Cell 119
(1), 47–60.
Garg, P., Burgers, P.M., 2005. DNA polymerases that propagate the eukaryotic DNA
replication fork. Crit. Rev. Biochem. Mol. Biol. 40 (2), 115–128.
Germond, J.E., Hirt, B., Oudet, P., Gross-Bellark, M., Chambon, P., 1975. Folding of the DNA
double helix in chromatin-like structures from simian virus 40. Proc. Natl. Acad. Sci.
U. S. A. 72 (5), 1843–1847.
Giacherio, D., Hager, L.P., 1979. A poly(dT)-stimulated ATPase activity associated with
simian virus 40 large T antigen. J. Biol. Chem. 254 (17), 8113–8116.
Gomez-Llorente, Y., Fletcher, R.J., Chen, X.S., Carazo, J.M., San Martin, C., 2005.
Polymorphism and double hexamer structure in the archaeal minichromosome
maintenance (MCM) helicase from Methanobacterium Thermoautotrophicum. J. Biol.
Chem. 280 (49), 40909–40915.
Griffith, J.D., 1975. Chromatin structure: deduced from a minichromosome. Science 187
(4182), 1202–1203.
Harlow, E., Crawford, L.V., Pim, D.C., Williamson, N.M., 1981. Monoclonal antibodies
specific for simian virus 40 tumor antigens. J. Virol. 39 (3), 861–869.
Hennessy, F., Nicoll, W.S., Zimmermann, R., Cheetham, M.E., Blatch, G.L., 2005. Not all J
domains are created equal: implications for the specificity of Hsp40–Hsp70
interactions. Protein Sci. 14 (7), 1697–1709.
Hubscher, U., Stalder, H.P., 1985. Mammalian DNA helicase. Nucleic Acids Res. 13 (15),
5471–5483.
358
Minireview
Indiani, C., O, Donnell, M., 2006. The replication clamp-loading machine at work in the
three domains of life. Nat. Rev. Mol. Cell Biol. 7 (10), 751–761.
Ishov, A.M., Maul, G.G., 1996. The periphery of nuclear domain 10 (ND10) as site of DNA
virus deposition. J. Cell Biol. 134 (4), 815–826.
Jacob, F., Brenner, S., 1963. [On the regulation of DNA synthesis in bacteria: the
hypothesis of the replicon.]. C. R. Hebdo. Seances Acad. Sci. 256, 298–300.
Jiang, X., Klimovich, V., Arunkumar, A.I., Hysinger, E.B., Wang, Y., Ott, R.D., Guler, G.D.,
Weiner, B., Chazin, W.J., Fanning, E., 2006. Structural mechanism of RPA loading on
DNA during activation of a simple pre-replication complex. EMBO J. 25 (23),
5516–5526.
Joo, W.S., Kim, H.Y., Purviance, J.D., Sreekumar, K.R., Bullock, P.A., 1998. Assembly of
T-antigen double hexamers on the simian virus 40 core origin requires only a
subset of the available binding sites. Mol. Cell. Biol. 18 (5), 2677–2687.
Kim, H.-Y., Ahn, B.-Y., Cho, Y., 2001. Structural basis for the inactivation of retinoblastoma
tumor suppressor by SV40 large T antigen. EMBO J. 20 (1), 295–304.
Kitamura, E., Blow, J.J., Tanaka, T.U., 2006. Live-cell imaging reveals replication of
individual replicons in eukaryotic replication factories. Cell 125 (7), 1297–1308.
Lavin, M.F., 2008. Ataxia-telangiectasia: from a rare disorder to a paradigm for cell
signalling and cancer. Nat. Rev. Mol. Cell Biol. 9 (10), 759–769.
Li, J.J., Kelly, T.J., 1984. Simian virus 40 DNA replication in vitro. Proc. Natl. Acad. Sci. U. S. A.
81 (22), 6973–6977.
Li, D., Zhao, R., Lilyestrom, W., Gai, D., Zhang, R., DeCaprio, J.A., Fanning, E., Jochimiak, A.,
Szakonyi, G., Chen, X.S., 2003. Structure of the replicative helicase of the
oncoprotein SV40 large tumour antigen. Nature 423 (6939), 512–518.
Luo, X., Sanford, D.G., Bullock, P.A., Bachovchin, W.W., 1996. Solution structure of the
origin DNA-binding domain of SV40 T-antigen. Nat. Struct. Biol. 3 (12),
1034–1039.
Margolskee, R.F., Nathans, D., 1984. Simian virus 40 mutant T antigens with relaxed
specificity for the nucleotide sequence at the viral DNA origin of replication. J. Virol.
49 (2), 386–393.
Mastrangelo, I.A., Hough, P.V., Wall, J.S., Dodson, M., Dean, F.B., Hurwitz, J., 1989. ATPdependent assembly of double hexamers of SV40 T antigen at the viral origin of
DNA replication. Nature 338 (6217), 658–662.
Matsumoto, T., Eki, T., Hurwitz, J., 1990. Studies on the initiation and elongation
reactions in the simian virus 40 DNA replication system. Proc. Natl. Acad. Sci. U. S. A.
87 (24), 9712–9716.
Meinke, G., Bullock, P.A., Bohm, A., 2006. Crystal structure of the simian virus 40 large
T-antigen origin-binding domain. J. Virol. 80 (9), 4304–4312.
Meinke, G., Phelan, P., Moine, S., Bochkareva, E., Bochkarev, A., Bullock, P.A., Bohm, A.,
2007. The crystal structure of the SV40 T-antigen origin binding domain in complex
with DNA. PLoS Biol. 5 (2), e23.
Meister, P., Taddei, A., Gasser, S.M., 2006. In and out of the replication factory. Cell 125
(7), 1233–1235.
Moarefi, I.F., Small, D., Gilbert, I., Hopfner, M., Randall, S.K., Schneider, C., Russo, A.A.,
Ramsperger, U., Arthur, A.K., Stahl, H., et al., 1993. Mutation of the cyclin-dependent
kinase phosphorylation site in simian virus 40 (SV40) large T antigen specifically
blocks SV40 origin DNA unwinding. J. Virol. 67 (8), 4992–5002.
Morrow, J.F., Berg, P., 1972. Cleavage of simian virus 40 DNA at a unique site by a
bacterial restriction enzyme. Proc. Natl. Acad. Sci. U. S. A. 69 (11), 3365–3369.
Moyer, S.E., Lewis, P.W., Botchan, M.R., 2006. Isolation of the Cdc45/Mcm2-7/GINS
(CMG) complex, a candidate for the eukaryotic DNA replication fork helicase. Proc.
Natl. Acad. Sci. U. S. A. 103 (27), 10236–10241.
Nathans, D., 1978. Restriction endonucleases, simian virus 40, and the new genetics.
Nobel Lecture. (December 8, 1978).
Nathans, D., Danna, K.J., 1972. Specific origin in SV40 DNA replication. Nat. New Biol. 236
(68), 200–202.
Ott, R.D., Rehfuess, C., Podust, V.N., Clark, J.E., Fanning, E., 2002. Role of the p68 subunit
of human DNA polymerase alpha-primase in simian virus 40 DNA replication. Mol.
Cell. Biol. 22 (16), 5669–5678.
Otto, B., Fanning, E., 1978. DNA polymerase alpha is associated with replicating SV40
nucleoprotein complexes. Nucleic Acids Res. 5 (5), 1715–1728.
Pascal, J.M., O, Brien, P.J., Tomkinson, A.E., Ellenberger, T., 2004. Human DNA ligase I
completely encircles and partially unwinds nicked DNA. Nature 432 (7016),
473–478.
Pospiech, H., Kursula, I., Abdel-Aziz, W., Malkas, L., Uitto, L., Kastelli, M., Vihinen-Ranta, M.,
Eskelinen, S., Syvaoja, J.E., 1999. A neutralizing antibody against human DNA
polymerase epsilon inhibits cellular but not SV40 DNA replication. Nucleic Acids Res.
27 (19), 3799–3804.
Reddy, V.B., Thimmappaya, B., Dhar, R., Subramanian, K.N., Zain, B.S., Pan, J., Ghosh, P.K.,
Celma, M.L., Weissman, S.M., 1978. The genome of simian virus 40. Science 200
(4341), 494–502.
Remus, D., Beall, E.L., Botchan, M.R., 2004. DNA topology, not DNA sequence, is a critical
determinant for Drosophila ORC-DNA binding. EMBO J. 23 (4), 897–907.
Roberts, R.J., 2005. How restriction enzymes became the workhorses of molecular
biology. Proc. Natl. Acad. Sci. U. S. A. 102 (17), 5905–5908.
Rossi, M.L., Pike, J.E., Wang, W., Burgers, P.M., Campbell, J.L.., Bambara, R.A., 2008. Pif1
helicase directs eukaryotic Okazaki fragments toward the two-nuclease cleavage
pathway for primer removal. J. Biol. Chem. 283 (41), 27483–27493.
Rundell, K., Collins, J.K., Tegtmeyer, P., Ozer, H.L., Lai, C.J., Nathans, D., 1977. Identification
of simian virus 40 protein A. J. Virol. 21 (2), 636–646.
Saragosti, S., Moyne, G., Yaniv, M., 1980. Absence of nucleosomes in a fraction of SV40
chromatin between the origin of replication and the region coding for the late
leader RNA. Cell 20 (1), 65–73.
Schwyzer, M., Weil, R., Frank, G., Zuber, H., 1980. Amino acid sequence analysis of
fragments generated by partial proteolysis from large simian virus 40 tumor
antigen. J. Biol. Chem. 255 (12), 5627–5634.
Sclafani, R.A., Holzen, T.M., 2007. Cell cycle regulation of DNA replication. Annu. Rev.
Genet. 41, 237–280.
Seki, T., Akita, M., Kamimura, Y., Muramatsu, S., Araki, H., Sugino, A., 2006. GINS is a DNA
polymerase epsilon accessory factor during chromosomal DNA replication in
budding yeast. J. Biol. Chem. 281 (30), 21422–21432.
Shi, Y., Dodson, G.E., Shaikh, S., Rundell, K., Tibbetts, R.S., 2005. Ataxia-telangiectasiamutated (ATM) is a T-antigen kinase that controls SV40 viral replication in vivo.
J. Biol. Chem. 280 (48), 40195–40200.
Shikata, K., Sasa-Masuda, T., Okuno, Y., Waga, S., Sugino, A., 2006. The DNA polymerase
activity of Pol epsilon holoenzyme is required for rapid and efficient chromosomal
DNA replication in Xenopus egg extracts. BMC Biochem. 7, 21.
Shortle, D., Nathans, D., 1978. Local mutagenesis: a method for generating viral mutants
with base substitutions in preselected regions of the viral genome. Proc. Natl. Acad.
Sci. U. S. A. 75 (5), 2170–2174.
Shortle, D.R., Margolskee, R.F., Nathans, D., 1979. Mutational analysis of the simian virus
40 replicon: pseudorevertants of mutants with a defective replication origin. Proc.
Natl. Acad. Sci. U. S. A. 76 (12), 6128–6131.
Simanis, V., Lane, D.P., 1985. An immunoaffinity purification procedure for SV40 large T
antigen. Virology 144 (1), 88–100.
Simmons, D.T., 2000. SV40 large T antigen functions in DNA replication and
transformation. Adv. Virus Res. 55, 75–134.
Smelkova, N.V., Borowiec, J.A., 1998. Synthetic DNA replication bubbles bound and
unwound with twofold symmetry by a simian virus 40 T-antigen double hexamer.
J. Virol. 72 (11), 8676–8681.
Stahl, H., Droge, P., Knippers, R., 1986. DNA helicase activity of SV40 large tumor antigen.
EMBO J. 5 (8), 1939–1944.
Staufenbiel, M., Deppert, W., 1983. Different structural systems of the nucleus are
targets for SV40 large T antigen. Cell 33 (1), 173–181.
Stillman, B., 1989. Initiation of eukaryotic DNA replication in vitro. Annu. Rev. Cell Biol. 5,
197–245.
Stillman, B.W., Gluzman, Y., 1985. Replication and supercoiling of simian virus 40 DNA in
cell extracts from human cells. Mol. Cell. Biol. 5 (8), 2051–2060.
Sullivan, C.S., Pipas, J.M., 2002. T antigens of simian virus 40: molecular chaperones for
viral replication and tumorigenesis. Microbiol. Mol. Biol. Rev. 66 (2), 179–202.
Tang, Q., Bell, P., Tegtmeyer, P., Maul, G.G., 2000. Replication but not transcription of
simian virus 40 DNA is dependent on nuclear domain 10. J. Virol. 74 (20), 9694–9700.
Tegtmeyer, P., 1972. Simian virus 40 deoxyribonucleic acid synthesis: the viral replicon.
J. Virol. 10 (4), 591–598.
Tegtmeyer, P., Ozer, H.L., 1971. Temperature-sensitive mutants of simian virus 40:
infection of permissive cells. J. Virol. 8 (4), 516–524.
Tjian, R., 1978. The binding site on SV40 DNA for a T antigen-related protein. Cell 13 (1),
165–179.
Tooze, J., 1973. The Molecular Biology of Tumour Viruses. Cold Spring Harbor
Laboratory, N.Y.
Tsurimoto, T., Stillman, B., 1991. Replication factors required for SV40 DNA replication in
vitro. II. Switching of DNA polymerase alpha and delta during initiation of leading
and lagging strand synthesis. J. Biol. Chem. 266 (3), 1961–1968.
Tsurimoto, T., Melendy, T., Stillman, B., 1990. Sequential initiation of lagging and leading
strand synthesis by two different polymerase complexes at the SV40 DNA
replication origin. Nature 346 (6284), 534–539.
Valle, M., Gruss, C., Halmer, L., Carazo, J.M., Donate, L.E., 2000. Large T-antigen double
hexamers imaged at the simian virus 40 origin of replication. Mol. Cell. Biol. 20 (1), 34–41.
Valle, M., Chen, X.S., Donate, L.E., Fanning, E., Carazo, J.M., 2006. Structural basis for the
cooperative assembly of large T antigen on the origin of replication. J. Mol. Biol. 357
(4), 1295–1305.
Vinograd, J., Lebowitz, J., Radloff, R., Watson, R., Laipis, P., 1965. The twisted circular form
of polyoma viral DNA. Proc. Natl. Acad. Sci. U. S. A. 53 (5), 1104–1111.
Virshup, D.M., Russo, A.A., Kelly, T.J., 1992. Mechanism of activation of simian virus 40
DNA replication by protein phosphatase 2A. Mol. Cell. Biol. 12 (11), 4883–4895.
Waga, S., Stillman, B., 1994. Anatomy of a DNA replication fork revealed by
reconstitution of SV40 DNA replication in vitro. Nature 369 (6477), 207–212.
Waga, S., Stillman, B., 1998. The DNA replication fork in eukaryotic cells. Annu. Rev.
Biochem. 67, 721–751.
Waqar, M.A., Evans, M.J., Huberman, J.A., 1978. Effect of 2′,3′-dideoxythymidine-5′triphosphate on HeLa cell in vitro DNA synthesis: evidence that DNA polymerase
alpha is the only polymerase required for cellular DNA replication. Nucleic Acids
Res. 5 (6), 1933–1946.
Weil, R., 1963. The denaturation and the renaturation of the DNA of polyoma virus. Proc.
Natl. Acad. Sci. U. S. A. 49, 480–487.
Weil, R., Vinograd, J., 1963. The cyclic helix and cyclic coil forms of polyoma viral DNA.
Proc. Natl. Acad. Sci. U. S. A. 50, 730–738.
Weinberg, D.H., Collins, K.L., Simancek, P., Russo, A., Wold, M.S., Virshup, D.M., Kelly, T.J.,
1990. Reconstitution of simian virus 40 DNA replication with purified proteins.
Proc. Natl. Acad. Sci. U. S. A. 87 (22), 8692–8696.
Weisshart, K., Bradley, M.K., Weiner, B.M., Schneider, C., Moarefi, I., Fanning, E., Arthur,
A.K., 1996. An N-terminal deletion mutant of simian virus 40 (SV40) large T antigen
oligomerizes incorrectly on SV40 DNA but retains the ability to bind to DNA
polymerase alpha and replicate SV40 DNA in vitro. J. Virol. 70 (6), 3509–3516.
Weisshart, K., Taneja, P., Jenne, A., Herbig, U., Simmons, D.T., Fanning, E., 1999. Two
regions of simian virus 40 T antigen determine cooperativity of double-hexamer
assembly on the viral origin of DNA replication and promote hexamer interactions
during bidirectional origin DNA unwinding. J. Virol. 73 (3), 2201–2211.
Weisshart, K., Friedl, S., Taneja, P., Nasheuer, H.-P., Schlott, B., Grosse, F., Fanning, E.,
2004. Partial proteolysis of simian virus 40 T antigen reveals intramolecular
contacts between domains and conformation changes upon hexamer assembly.
J. Biol. Chem. 279 (37), 38943–38951.
Minireview
Wessel, R., Schweizer, J., Stahl, H., 1992. Simian virus 40 T-antigen DNA helicase is a
hexamer which forms a binary complex during bidirectional unwinding from the
viral origin of DNA replication. J. Virol. 66 (2), 804–815.
White, M., Eason, R., 1971. Nucleoprotein complexes in simian virus 40-infected cells.
J. Virol. 8 (4), 363–371.
Wobbe, C.R., Dean, F., Weissbach, L., Hurwitz, J., 1985. In vitro replication of duplex
circular DNA containing the simian virus 40 DNA origin site. Proc. Natl. Acad. Sci.
U. S. A. 82 (17), 5710–5714.
Wu, X., Avni, D., Chiba, T., Yan, F., Zhao, Q., Lin, Y., Heng, H., Livingston, D., 2004. SV40 Tantigen
interacts with Nbs1 to disrupt DNA replication control. Genes Dev. 18 (11), 1305–1316.
359
Yuzhakov, A., Kelman, Z., Hurwitz, J., O, Donnell, M., 1999. Multiple competition
reactions for RPA order the assembly of the DNA polymerase delta holoenzyme.
EMBO J. 18 (21), 6189–6199.
Zhao, X., Madden-Fuentes, R.J., Lou, B.X., Pipas, J.M., Gerhardt, J., Rigell, C.J., Fanning, E.,
2008. Ataxia telangiectasia-mutated damage-signaling kinase- and proteasomedependent destruction of Mre11-Rad50-Nbs1 subunits in simian virus 40-infected
primate cells. J. Virol. 82 (11), 5316–5328.
Zlotkin, T., Kaufmann, G., Jiang, Y., Lee, M.Y., Uitto, L., Syvaoja, J., Dornreiter, I., Fanning, E.,
Nethanel, T., 1996. DNA polymerase epsilon may be dispensable for SV40- but not
cellular-DNA replication. EMBO J. 15 (9), 2298–2305.