Minireview

SV40 DNA replication: From the A gene to a nanomachine

Ellen Fanning !, Kun ZhaoDepartment of Biological Sciences and Vanderbilt-Ingram Cancer Center, Vanderbilt University, VU Station B 351634, Nashville, TN 37235-1634, USA

a b s t r a c ta r t i c l e i n f o

Article history:Received 11 November 2008Accepted 18 November 2008Available online 20 December 2008

Keywords:Genome structureDNA replication originT antigenHelicaseDouble hexamerReplication forkHand-offPolymerase switchDNA damage signaling

Duplication of the simian virus 40 (SV40) genome is the best understood eukaryotic DNA replication processto date. Like most prokaryotic genomes, the SV40 genome is a circular duplex DNA organized in a singlereplicon. This small viral genome, its association with host histones in nucleosomes, and its dependence onthe 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 initiallyde!ned using a cell-free SV40 replication reaction. Although our understanding of the vastly more complexhost replication fork is advancing, no eukaryotic replisome has yet been reconstituted and the SV40 paradigmremains a point of reference. This article reviews some of the milestones in the development of this paradigmand 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 laidthe foundations of modern molecular biology. The physicists,chemists, biologists, and physicians pioneered this frontier with thehope that the relative simplicity of these agents might allow them toserve as tools to understand their vastly more complex infected hostcells. The discovery of simple DNA viruses that propagated inmammalian cell nuclei and caused tumors in experimental animalsled the way to an explosion of eukaryotic molecular biology and itsapplications to understanding, treating, and preventing humandisease. For pioneering studies of the DNA tumor viruses polyomavirusand SV40 in the 1969's, Renato Dulbecco was awarded the 1975 NobelPrize in Physiology or Medicine. Equally importantly, the many youngscientists who trained in his laboratory were inspired to pursue andexpand on this fruitful approach in ever more exciting new directions.The development of the !eld and collegial interactions amongmembers of the DNA tumor virus community were greatly fosteredby annual meetings sponsored by Cold Spring Harbor Laboratory andImperial Cancer Research Fund, as well as by review volumes edited byJohn Tooze beginning in 1973 (Tooze, 1973).

This article reviews some of the fundamental lessons on genomestructure, DNA replication, and genome maintenance that thesedeceptively simple viruses have revealed over the past 4 decades.The utility of these viral paradigms in guiding the investigation ofmammalian DNA replication is considered. The article concludes with

re"ections on how the rapidly growing understanding of host genomemaintenance is leading to a re-consideration of how these virusesexploit their host cells.

The SV40 minichromosome: genetic and physical maps linkedthrough DNA sequence

The SV40 genome is a covalently closed circular duplex DNAmolecule of 3.6!106 Da (Crawford and Black,1964; Dulbecco and Vogt,1963; Weil and Vinograd, 1963). Biophysical characterization ofsuperhelical SV40 and polyomavirus DNA provided the !rst insightinto the initially puzzling ability of supercoiledDNA to renature rapidlyafter exposure to alkali (Vinograd et al., 1965; Weil, 1963; Weil andVinograd, 1963), its limited uptake of intercalating dyes, e.g. ethidiumbromide, and other properties typical of supercoiled DNA. The SV40genome exists in the virus particle and in infected cells as a mini-chromosome packaged with host cell histones into nucleosomes thatclosely resemble those of the host chromatin (Bonner et al., 1968;Germond et al., 1975; Grif!th, 1975; White and Eason, 1971) (Fig. 1).Puri!cation of SV40DNA fromminichromosomes reveals its negativelysupercoiled topology. As we now know, this topology endowschromatin with the capacity to readily denature for initiation of DNAreplication or transcription.

Genetic studies of replication in prokaryotes had led to apotentially general model for control of replication: the repliconmodel of Jacob, Brenner, and Cuzin (Jacob and Brenner, 1963). Themodel postulated a cis-acting element, the replicator, recognized by atrans-acting factor, the initiator. This interaction would lead to locallydenatured duplex DNA in or near the replicator element and initiation

Virology 384 (2009) 352–359

! Corresponding author. Fax: +1 615 343 6707.E-mail address: ellen.fanning@vanderbilt.edu (E. Fanning).

0042-6822/$ – see front matter © 2008 Elsevier Inc. All rights reserved.doi:10.1016/j.virol.2008.11.038

Contents lists available at ScienceDirect

Virology

j ourna l homepage: www.e lsev ie r.com/ locate /yv i ro

of replication. Each replicator with its initiator would thus govern thereplication of the "anking regions of DNA, the replicon. If this modelwere general, one might expect eukaryotic DNA to be organized intoreplicons in a similar manner. If SV40 DNA represents a eukaryoticreplicon, one would predict a genetically de!nable viral replicatorelement and an initiator that recognized it.

In the mind of Daniel Nathans at Johns Hopkins University, theappeal of SV40 as an object for genetic analysis converged with thediscovery of the !rst sequence-speci!c restriction endonucleases byhis colleague Hamilton Smith (Fig. 2). Nathans and colleaguesgenerated the !rst restriction cleavage map of SV40 DNA, bydetermining the physical order of the Hind II/III and Hpa I/II sitesaround 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 pulse-chase approach with their restriction map to determine the physicalstart site for SV40 DNA replication, the origin, and show thatreplication proceeded bidirectionally to terminate on the oppositeside of the DNA molecule (Danna and Nathans, 1972; Nathans andDanna, 1972). [For a fascinating overview of these discoveries, seeBrownlee, 2005; Roberts, 2005] This physical map greatly facilitateddetermination of the 5243 bp sequence of SV40 DNA, the !rsteukaryotic genome to be completely sequenced (Fiers et al., 1978;Reddy et al., 1978). Moreover, the map and the sequence enabled theclassical mutational analysis of the SV40 genome (Chou and Martin,1974; Tegtmeyer, 1972; Tegtmeyer and Ozer, 1971) to be correlatedwith nucleotide sequence changes that affected viral DNA replication(temperature-sensitive complementation group A (tsA)), cell trans-formation (tsA), and virion production (tsB, C, BC, D) [for a personalaccount, see Nathans, 1978].

With the viral DNA sequence in hand and new restrictionendonucleases rapidly emerging in several laboratories, the Nathanslab moved quickly to test the function of the SV40 origin of DNAreplication by mutational analysis. They devised site-directedmutagenesis protocols for deletions and base substitutions followedby selection for resistance to cleavage by Bgl I, which has a singlerecognition site in SV40 DNA at the origin (DiMaio and Nathans, 1980;DiMaio and Nathans, 1982; Shortle and Nathans, 1978). Thesemutations were mapped by DNA sequencing and shown to renderSV40 replication defectivewhen the genomewas introduced into hostcells, satisfying one criterion for a replicator element. To examine therelationship between the putative replicator element and the tsA genethat was also involved in replication, the Nathans lab carried out amutational screen for second site revertants of the replication-

defective mutant origins. These pseudorevertant mutations werethen mapped and shown to reside at positions outside of the originregion and to alter the coding sequence of the tsA gene (Margolskeeand Nathans, 1984; Shortle et al., 1979). The A gene encodes the SV40large tumor (T) antigen (Tag), a multifunctional protein whosestructure and roles in viral DNA replication are reviewed below.Thus, the origin element interacted genetically with a viral gene thatregulated the rate of viral DNA replication, providing strong evidencefor a replicon model in controlling replication of SV40 DNA.Biochemical investigation of Tag promptly con!rmed the interaction,paving the way for new experiments to elucidate the mechanism ofSV40 DNA replication.

Further dissection of the viral replicator in multiple laboratoriesrevealed a 64 bp core composed of three elements. A central elementcontains a palindromic array of four GAGGC pentanucleotides that, aswe now know, serve as binding sites for Tag. The binding sites are"anked by an easily denatured imperfect palindrome (EP) on one sideand by an AT-rich sequence on the other side. The two "ankingelements undergo local distortion or melting during initiation ofreplication (Borowiec et al., 1990). The early and late promoterelements "ank the viral core origin and stimulate its activity ininfected cells, as does the viral enhancer element. These auxiliaryelements may stimulate initiation of replication from the viral coreorigin at multiple levels, some of which may re"ect the closerelationship between origins of replication and transcription. The!rst level may be by modulating the structure of the core origin DNAto facilitate distortion by Tag, e.g. through intrinsically bent AT-richDNA sequences, or local modulation of supercoiling. These physicalproperties of origin DNA are also found in eukaryotic chromosomalorigins of replication and are thought to be important for binding ofthe origin recognition complex ORC (Remus et al., 2004). Proteinsbound to the auxiliary elements may also facilitate Tag assembly onthe core origin DNA, Tag remodeling into an active helicase, orrecruitment of host replication proteins. For example, chromatinremodeling to generate a nucleosome-free origin region and histonemodi!cations are likely to be important for initiation at the SV40origin and at chromosomal origins (Saragosti et al., 1980). Lastly,replication of the viral minichromosome appears to take place inspeci!c subnuclear domains (Ishov and Maul, 1996; Staufenbiel andDeppert, 1983; Tang et al., 2000), but how the minichromosome istargeted to these sites remains poorly de!ned.

Given the sequence speci!city of the SV40 core origin and thedependence of the virus on host cell proteins for DNA replication, itwas tempting to imagine that chromosomal origins of replicationmight also be composed of modules with de!ned sequences that

Fig. 1. Electron micrograph of an SV40 minichromosome isolated from productivelyinfected cells. (Scale bar 100 nm). (Reprinted from Grif!th, 1975 with permissionfrom AAAS.)

Fig. 2. Daniel Nathans (left) and Hamilton Smith in the laboratory at Johns HopkinsUniversity. (Reprinted from Roberts, 2005 with permission from Copyright 2005National Academy of Sciences, U.S.A.)

353Minireview

could be recognized by initiator proteins. Although origins in buddingyeast ful!ll this expectation to some extent, no common consensussequence has been found in either the genetically de!ned replicatorsor 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 DNAreplication

Tag was !rst detected as a 90–100 kDa polypeptide from infectedcell extracts that reacted with the serum of rodents bearing tumorsinduced by SV40 injection (Rundell et al., 1977). Initially it wasuncertain whether Tag was in fact encoded by the A gene in the SV40genome since small deletions in that gene failed to reduce theapparent mass of the immunoreactive protein. This conundrum wasresolved through the combined efforts of the Tegtmeyer, Crawford andBerg labs (Fig. 3) (Crawford et al., 1978), providing the !rst hint thatTag was expressed from one of the three alternatively spliced earlySV40 transcripts (see Yaniv article in this issue).

Taking advantage of the high level expression of a Tag-relatedadenovirus-SV40 hybrid protein D2, Robert Tjian succeeded in aclassical puri!cation of the !rst native, biochemically active form ofTag (Tjian, 1978). This key achievement was the !rst step towardde!ning the mechanism of SV40 DNA replication. Tjian showed thatpuri!ed D2 bound to DNA and was capable of speci!cally protectingSV40 origin DNA sequences against nuclease digestion, con!rming thegenetic interactions between the SV40 origin and the A gene encodingTag. Moreover, D2 bound sequentially to several elements of the viralorigin DNA in a manner suggesting possible multimerization of theprotein on the DNA to protect up to 120 bp. Subsequent work inmultiple laboratories led to de!nition of the pentanucleotide GAGGCas the fundamental recognition motif speci!cally bound by Tag, eitheras 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 4pentanucleotides separated by 1 bp (site II) (reviewed by Borowiecet al., 1990; Challberg and Kelly, 1989; Fanning and Knippers, 1992;Stillman, 1989).

Puri!ed D2 and Tag were soon shown to display a secondbiochemical 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 Tagmight have DNA helicase activity, it could not be convincinglydemonstrated.Using Tagpuri!ed from infected cells by immunoaf!nitychromatography 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), theKnippers lab showed that highly puri!ed Tag could unwind partial

duplex DNA with 3" to 5" polarity in an ATP-hydrolysis dependentmanner (Fig. 4) (Stahl et al., 1986). Moreover, monoclonal antibodiesagainst speci!c regions of Tag inhibited helicase activity andmutationsin Tag that reduced ATPase activity also reduced helicase activity (Stahlet al.,1986). The helicase activity of Tagwasquickly con!rmed in severalother laboratories (reviewed in Fanning and Knippers, 1992).

Despite this important step in understanding the role of Tag in viralDNA replication, it remained unclear how the helicase activity of Tagcould unwind duplex DNA from the origin. The Hurwitz laboratory,collaborating with several others, discovered that Tag assembles into amultimer on the viral origin in an ATP-binding dependent manner toform a bilobed double hexameric structure that distorts the duplexDNA locally (Borowiec et al., 1990; Dodson et al., 1987; Mastrangeloet al., 1989). In the presence of Mg-ATP, a single-stranded DNA bindingprotein, and topoisomerase I, Tag generated a theta-like structurewithsingle-stranded bubbles of varying sizes with the origin always at thecenter of the bubble (Dean et al., 1987a; Dean et al., 1987b; Dodsonet al., 1987). A large protein complex likely to be Tag hexamer wasoften observed at the junctions of the single-stranded bubble withduplex DNA, suggesting that two diverging Tag hexamers unwoundthe duplex at a similar rate. A few years later, under differentexperimental conditions, active unwinding complexes of doublehexamer were visualized with two loops of ssDNA emanating fromthe double hexamer (Fig. 5). These images suggested a moresophisticated bidirectional unwinding mechanism in which duplexparental DNA is reeled into the double hexamer, coordinately fromboth sides, and the unwound template is spooled out for replication(Wessel et al., 1992). This type of unwinding intermediate, notobserved in initiation of prokaryotic replication by 5" to 3" helicases(Fang et al., 1999), implied some kind of functional contacts betweenTag hexamers. Biochemical, genetic, and structural data (Meinke et al.,2006; Moare! 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 102–259in one hexamer with the corresponding residues in the otherhexamer, strongly supporting this model of bidirectional unwindingas physiologically relevant (reviewed in Bullock, 1997; Fanning, 1994;

Fig. 3. Peter Tegtmeyer (left) in discussion at a DNA Tumor Virus meeting, University ofWisconsin-Madison (Courtesy of K. Rundell).

Fig. 4. Rolf Knippers in his laboratory at the University of Konstanz, Germany, in the1980's. (Courtesy of M. Baack).

354 Minireview

Simmons, 2000). This model suggests that both replication forks mayassemble, at least initially, on the Tag double hexamer and thatprogression 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 laboratoriesembarked on a major quest to establish a cell-free SV40 DNA systemthat could be used to identify the host proteins necessary to replicateviral DNA. Early work with replicating SV40 nucleoprotein complexesisolated from infected cells had already been shown to completereplication in cell-free extracts (DePamphilis et al., 1975; Edenberget al., 1976) and DNA polymerase alpha-primase had been identi!ed as

a key activity (Otto and Fanning, 1978; Waqar et al., 1978). Joachim Liand Thomas Kelly (1984)were the !rst to succeed in using puri!edSV40 origin DNA and extracts from primate cells supplemented withimmunopuri!ed Tag to replicate a DNA template from the viral origin,followed quickly by independent studies in the Stillman and Hurwitzlaboratories (Li and Kelly, 1984; Stillman and Gluzman, 1985; Wobbeet al., 1985) (Figs. 6, 7). Countless hours of painstaking work in coldrooms in these and other labs resulted in the puri!cation of ten humanproteins that, together with Tag, are suf!cient to reconstitute thereplication 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 forindividual steps were reconstituted with puri!ed proteins. Threestages of the replication process have been reconstituted and studiedin detail: initiation, elongation, and Okazaki fragment maturation.Four proteins (Tag, RPA, DNA polymerase alpha-primase, andtopoisomerase I) are suf!cient to reconstitute initiation of replicationat the viral origin (Matsumoto et al., 1990) reviewed by Borowiec et al.,(1990) and Bullock, (1997). After primer synthesis, the clamp-loaderreplication factor C (RFC) orchestrates a switch from DNA polymerasealpha-primase to the more processive DNA polymerase delta(Tsurimoto et al., 1990; Tsurimoto and Stillman, 1991; Weinberget al., 1990) (Fig. 8). RPA contacts with RFC appear to play a role indisplacing DNA polymerase alpha-primase from the template(Yuzhakov et al., 1999). RFC bound to ATP binds the sliding clampPCNA, cracks open the ring, and RFC contact with primer-templatetriggers ATP hydrolysis, releasing a closed PCNA complex loaded at theprimer-terminus and able to bind and position polymerase delta forprimer extension (Indiani and O'Donnell, 2006).

Despite the simplicity of the SV40 replication fork and several keydifferences relative to host replication forks and their intricateregulatory wiring, the SV40 fork continues to serve as a usefulparadigm for host forks (Figs. 7, 8), where new challenges await.Structures of many of these host replication proteins and theirdomains have now been determined by X-ray crystallography (forexamples, see Bowman et al., 2004; Fanning et al., 2006; Garg andBurgers, 2005; Indiani and O'Donnell, 2006; Pascal et al., 2004),opening the possibility of developing an atomic level understanding ofreplication fork operation in eukaryotes. De!nition of how theseexchanges of proteins and coupling of leading and lagging strandreplication are accomplished will require more complete structuralinformation on DNA polymerases, de!nition of protein interactionsrequired for hand-off reactions, and single molecule studies.

Seeing is believing: structures of SV40 Tag

Mapping of functional domains of Tag by molecular genetics andbiochemistry provided the !rst hints that SV40 Tag is a multi-domain

Fig. 6. Thomas J. Kelly (left) and Bruce Stillman (right) at a reception during the 1994Cold Spring Harbor Symposium (Courtesy of Cold Spring Harbor Archives).

Fig. 7. Aminimal set of replicationproteins at a eukaryotic fork. MCMhelicase substituteshere for Tag; the PCNA clamp loader RFC and topoisomerases are not shown. (Reprintedfrom 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 wasincubated under unwinding conditions with a duplex plasmid DNA fragment contain-ing the SV40 origin 1.1 or 1.5 kb from the ends. The reaction was terminated byglutaraldehyde, the sample was puri!ed, spread, negatively stained, and visualized byelectron microscopy. Two single-stranded loops bound to single-stranded DNA bindingprotein emanate from a Tag double hexamer (see inset). (Scale bar 100 nm). The doublehexamer 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 1992American Society for Microbiology.)

355Minireview

protein with a bewildering number of functions (Fanning, 1992).Mapping of structured domains by limited proteolysis correlated tosome extent with the functional domain map (Schwyzer et al., 1980),but an atomic structure of Tag remained elusive until the focus shiftedto 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 withpreviously mapped mutations that affected origin DNA binding,provided important insight into the mechanism of origin recognitionby Tag and assembly of the double hexamer (Bullock, 1997; Joo et al.,1998). The next domain to come into view was the DnaJ co-chaperonedomain (Fig. 9) in complex with a fragment of the retinoblastomatumor suppressor protein (Kimet al., 2001; reviewedbyHennessyet al.,2005; Sullivan and Pipas, 2002). Although the DnaJ function is not

required for SV40 replication in cell-free reactions (Weisshart et al.,1996), its functions are essential for DNA replication in productivelyinfected cells (Campbell et al.,1997). In a keyadvance for understandingthe mechanism of initiation of viral DNA replication, the !rst crystalstructure of the Tag helicase domain was determined in XiaojiangChen'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). Theorigin DNA binding domain and the DnaJ domain are tethered to oneanother and to the helicase domain through "exible linkers (Fig. 9).The structure of the host range domain (residues 628–708) remainsunknown (see article by Pipas in this issue). These structured domainscorrelate very well with the functional domains of Tag deduced bybiochemistry and molecular genetics (see Fanning and Knippers,1992; Weisshart et al., 2004). Remarkably, the Tag helicase structure isclosely related to that of the chromosomal replicative helicase, asevidenced by crystal structures of archaeal MCM helicase domains(Fletcher et al., 2003; Gomez-Llorente et al., 2005; reviewed bySclafani and Holzen, 2007).

The Tag structures available so far suggest a modular molecule thatlikely operates dynamically to coordinate origin DNA binding andlocal distortion by the double hexamer, and re-organization of thedouble hexamer into a bidirectional unwinding machine (Bochkarevaet al., 2006; Gai et al., 2004; Meinke et al., 2006; Meinke et al., 2007;Valle et al., 2006). Re-examination of the initiation reaction andmapping of protein–protein interactions at the atomic level isbeginning to provide insight into the dynamic nature of the initiationprocess. During initiation of replication, RPA associationwith Tag-OBDcouples origin DNA unwinding to loading of RPA on the emergingtemplate (Jiang et al., 2006). Similarly, Tag-OBD-mediated remodelingof RPA-ssDNA complexes facilitates the ssDNA hand-off to the DNApolymerase alpha-primase positioned on the Tag helicase domain(Arunkumar et al., 2005). Nevertheless, until the relative positions ofthe Tag domains in the double hexamer and the path of the DNAtemplate through the protein during various functional states ofreplication can be visualized, our understanding of the SV40replication process is incomplete. One successful path toward thisultimate goal began by analyzing electron micrographs of Tag doublehexamers on origin DNA with image processing algorithms (Valleet al., 2000). Recent advances in cryo-electronmicroscopy of wild typeand mutant Tag on origin DNA, new image classi!cation algorithms,and !tting of new atomic structures of Tag may allow this goal to bereached.

The SV40 replication paradigm: a perspective

Much effort has been aimed at elucidating in detail the operationof the SV40 replisome with the hope that general principles can be

Fig. 9. Modular organization of Tag domains and linkers. Atomic structures of DnaJ (Kimet 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.)

Fig. 10. Crystallographer Xiaojiang S. Chen, University of Southern California.

Fig. 8. Linking the polymerase switch to Okazaki fragment processing. The mechanismof elongation of a primed DNA template on the leading strand or for each Okazakifragment on the lagging strand was studied in reactions reconstituted with puri!edproteins (steps 1–4). The mechanism of Okazaki fragment maturation was alsoelucidated in reconstituted reactions (steps 5–7). (Reprinted from Waga and Stillman,1998 with permission from Annual Reviews.) (For a current view of Okazaki fragmentprocessing, see Rossi et al., 2008).

356 Minireview

discerned that will increase our understanding of mammalianchromosomal replication. The bidirectional “replication factory”organization of the SV40 replisome appears to be a simple exampleof those that duplicate bacterial and eukaryotic chromosomes(Kitamura et al., 2006; Meister et al., 2006). However, SV40 and hostDNA replication mechanisms differ in several major features. SV40encodes its own DNA helicase, whereas chromosomal replicationdepends on the Cdc45/Mcm2-7/GINS helicase (Moyer et al., 2006 andreferences therein). DNA polymerase epsilon is important forchromosomal replication (Seki et al., 2006; Shikata et al., 2006), butSV40 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 phaseof the cell cycle, but DNA polymerase alpha-primase phosphorylatedby cyclin-dependent kinase is unable to support replication of viralDNA in a cell-free reaction (Ott et al., 2002 and references therein).Importantly, SV40 and polyomavirus infection induce DNA damagesignaling that promotes viral DNA replication (Dahl et al., 2005; Shiet al., 2005; Wu et al., 2004; Zhao et al., 2008), but ordinarily inhibitschromosomal replication and cell cycle transitions (Cimprich andCortez, 2008; Lavin, 2008). Because of these differences, one of themost critical open questions is whether the SV40 replisome operatesas a streamlined mimic of host chromosomal replication or rather, ahost DNA repair pathway. The unanticipated role of DNA damagesignaling in viral DNA replication raises the question of whether otherundiscovered factors and mechanisms might participate in SV40replication in infected cells, including the Hsc70 interaction with theTag DnaJ domain, sister cohesion and chromosome segregation,subnuclear positioning of viral genomes, interaction with theubiquitin-proteasome system, and chromatin modi!cation. TheSV40 model, with its rich history and wealth of experimental tools,may have an exciting future.

Acknowledgments

EF thanks her mentors, lab members, colleagues, collaborators, andfellow travelers along the SV40 road for fruitful cooperation, compa-nionship, guidance, and inspiration. B. Stillman, C. Clark, K. Rundell, X.S.Chen, and M. Baack kindly provided photographs. Financial supportfrom NIH (GM52948 to EF and P30 CA68485 to the Vanderbilt-IngramCancer Center) and Vanderbilt University are gratefully acknowledged.

References

Aladjem, M.I., 2007. Replication in context: dynamic regulation of DNA replicationpatterns in metazoans. Nat. Rev. Genet. 8 (8), 588–600.

Aladjem, M.I., Fanning, E., 2004. The replicon revisited: an old model learns new tricksin 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 simianvirus 40 replisome. Nat. Struct. Mol. Biol. 12 (4), 332–339.

Bell, S.P., 2002. The origin recognition complex: from simple origins to complexfunctions. Genes Dev. 16 (6), 659–672.

Bochkareva, E., Martynowski, D., Seitova, A., Bochkarev, A., 2006. Structure of the origin-binding 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. Thebiology 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 Tantigen 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 eukaryoticsliding DNA clamp-clamp loader complex. Nature 429 (6993), 724–730.

Brownlee, C., 2005. Danna and Nathans: restriction enzymes and the boon to modernmolecular 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 chaperonedomain of SV40 large T antigen promotes ef!cient viral DNA replication. GenesDev. 11 (9), 1098–1110.

Challberg, M.D., Kelly, T.J., 1989. Animal virus DNA replication. Annu. Rev. Biochem. 58,671–717.

Chou, J.Y.,Martin, R.G.,1974. Complementation analysis of simianvirus40mutants. 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 signalingpathway by polyomavirus. J. Virol. 79 (20), 13007–13017.

Danna, K., Nathans, D., 1971. Speci!c cleavage of simian virus 40 DNA by restrictionendonuclease of Hemophilus in!uenzae. 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. Acleavage 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 virus40 large tumor antigen requires three core replication origin domains for DNAunwinding 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 DNAcontaining 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 40deoxyribonucleic acid in cell lysates!. J. Biol. Chem. 250 (11), 4340–4347.

Deppert, W., Gurney, E.G., Harrison, R.O., 1981. Monoclonal antibodies against simianvirus 40 tumor antigens: analysis of antigenic binding sites, using adenovirus type2-simian virus 40 hybrid viruses. J. Virol. 37 (1), 478–482.

DiMaio, D., Nathans, D.,1980. Cold-sensitive regulatorymutants of simian virus 40. J. Mol.Biol. 140 (1), 129–142.

DiMaio, D., Nathans, D., 1982. Regulatory mutants of simian virus 40. Effect of mutationsat 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. Puri!cation of simian virus 40 large T antigen byimmunoaf!nity chromatography. J. Virol. 53 (3), 1001–1004.

Dodson, M., Dean, F.B., Bullock, P., Echols, H., Hurwitz, J., 1987. Unwinding of duplex DNAfrom 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 ofsimian 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 !xed double replisome.Trends Genet. 16 (2), 88–92.

Fang, L., Davey, M.J., O, Donnell, M., 1999. Replisome assembly at oriC, the replicationorigin of E. coli, reveals an explanation for initiation sites outside an origin. Mol. Cell4 (4), 541–553.

Fanning, E., 1992. Simian virus 40 large T antigen: the puzzle, the pieces, and theemerging picture. J. Virol. 66 (3), 1289–1293.

Fanning, E., 1994. Control of SV40 DNA replication by protein phosphorylation: a modelfor cellular DNA replication? Trends Cell Biol. 4 (7), 250–255.

Fanning, E., Knippers, R., 1992. Structure and function of simian virus 40 large tumorantigen. 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., VandeVoorde, A., VanHeuverswyn,H.,Van Herreweghe, J., Volckaert, G., Ysebaert, M., 1978. Complete nucleotide sequenceof 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. Thestructure 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 conformationalchange 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 DNAreplication 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 DNAdouble 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 withsimian 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 minichromosomemaintenance (MCM) helicase fromMethanobacterium Thermoautotrophicum. J. Biol.Chem. 280 (49), 40909–40915.

Grif!th, 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 antibodiesspeci!c 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 Jdomains are created equal: implications for the speci!city of Hsp40–Hsp70interactions. Protein Sci. 14 (7), 1697–1709.

Hubscher, U., Stalder, H.P., 1985. Mammalian DNA helicase. Nucleic Acids Res. 13 (15),5471–5483.

357Minireview

Indiani, C., O, Donnell, M., 2006. The replication clamp-loading machine at work in thethree 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 DNAvirus deposition. J. Cell Biol. 134 (4), 815–826.

Jacob, F., Brenner, S., 1963. [On the regulation of DNA synthesis in bacteria: thehypothesis 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 onDNA 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 ofT-antigen double hexamers on the simian virus 40 core origin requires only asubset 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 retinoblastomatumor 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 ofindividual replicons in eukaryotic replication factories. Cell 125 (7), 1297–1308.

Lavin, M.F., 2008. Ataxia-telangiectasia: from a rare disorder to a paradigm for cellsignalling and cancer. Nat. Rev. Mol. Cell Biol. 9 (10), 759–769.

Li, J.J., Kelly, T.J.,1984. Simian virus 40DNA 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 theoncoprotein SV40 large tumour antigen. Nature 423 (6939), 512–518.

Luo, X., Sanford, D.G., Bullock, P.A., Bachovchin, W.W., 1996. Solution structure of theorigin 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 relaxedspeci!city 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. ATP-dependent assembly of double hexamers of SV40 T antigen at the viral origin ofDNA replication. Nature 338 (6217), 658–662.

Matsumoto, T., Eki, T., Hurwitz, J., 1990. Studies on the initiation and elongationreactions 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 largeT-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 complexwith 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.

Moare!, 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-dependentkinase phosphorylation site in simian virus 40 (SV40) large T antigen speci!callyblocks 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 abacterial 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. Speci!c origin in SV40 DNA replication. Nat. NewBiol. 236(68), 200–202.

Ott, R.D., Rehfuess, C., Podust, V.N., Clark, J.E., Fanning, E., 2002. Role of the p68 subunitof 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 SV40nucleoprotein complexes. Nucleic Acids Res. 5 (5), 1715–1728.

Pascal, J.M., O, Brien, P.J., Tomkinson, A.E., Ellenberger, T., 2004. Human DNA ligase Icompletely 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 DNApolymerase 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 criticaldeterminant for Drosophila ORC-DNA binding. EMBO J. 23 (4), 897–907.

Roberts, R.J., 2005. How restriction enzymes became the workhorses of molecularbiology. 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. Pif1helicase directs eukaryotic Okazaki fragments toward the two-nuclease cleavagepathway 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. Identi!cationof 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 SV40chromatin between the origin of replication and the region coding for the lateleader RNA. Cell 20 (1), 65–73.

Schwyzer, M., Weil, R., Frank, G., Zuber, H., 1980. Amino acid sequence analysis offragments generated by partial proteolysis from large simian virus 40 tumorantigen. 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 DNApolymerase epsilon accessory factor during chromosomal DNA replication inbudding yeast. J. Biol. Chem. 281 (30), 21422–21432.

Shi, Y., Dodson, G.E., Shaikh, S., Rundell, K., Tibbetts, R.S., 2005. Ataxia-telangiectasia-mutated (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 polymeraseactivity of Pol epsilon holoenzyme is required for rapid and ef!cient chromosomalDNA replication in Xenopus egg extracts. BMC Biochem. 7, 21.

Shortle, D., Nathans, D., 1978. Local mutagenesis: a method for generating viral mutantswith 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 virus40 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 immunoaf!nity puri!cation procedure for SV40 large Tantigen. Virology 144 (1), 88–100.

Simmons, D.T., 2000. SV40 large T antigen functions in DNA replication andtransformation. Adv. Virus Res. 55, 75–134.

Smelkova, N.V., Borowiec, J.A., 1998. Synthetic DNA replication bubbles bound andunwound 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 aretargets 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 incell 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 forviral 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 ofsimian virus 40DNA 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 HarborLaboratory, N.Y.

Tsurimoto, T., Stillman, B., 1991. Replication factors required for SV40 DNA replication invitro. II. Switching of DNA polymerase alpha and delta during initiation of leadingand lagging strand synthesis. J. Biol. Chem. 266 (3), 1961–1968.

Tsurimoto, T., Melendy, T., Stillman, B., 1990. Sequential initiation of lagging and leadingstrand synthesis by two different polymerase complexes at the SV40 DNAreplication origin. Nature 346 (6284), 534–539.

Valle, M., Gruss, C., Halmer, L., Carazo, J.M., Donate, L.E., 2000. Large T-antigen doublehexamers imagedat the simianvirus 40origin 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 thecooperative 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 formof 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 40DNA replication by protein phosphatase 2A. Mol. Cell. Biol. 12 (11), 4883–4895.

Waga, S., Stillman, B., 1994. Anatomy of a DNA replication fork revealed byreconstitution 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 polymerasealpha is the only polymerase required for cellular DNA replication. Nucleic AcidsRes. 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 puri!ed proteins.Proc. Natl. Acad. Sci. U. S. A. 87 (22), 8692–8696.

Weisshart, K., Bradley, M.K., Weiner, B.M., Schneider, C., Moare!, I., Fanning, E., Arthur,A.K., 1996. An N-terminal deletion mutant of simian virus 40 (SV40) large T antigenoligomerizes incorrectly on SV40 DNA but retains the ability to bind to DNApolymerase 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. Tworegions of simian virus 40 T antigen determine cooperativity of double-hexamerassembly on the viral origin of DNA replication and promote hexamer interactionsduring 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 intramolecularcontacts between domains and conformation changes upon hexamer assembly.J. Biol. Chem. 279 (37), 38943–38951.

358 Minireview

Wessel, R., Schweizer, J., Stahl, H., 1992. Simian virus 40 T-antigen DNA helicase is ahexamer which forms a binary complex during bidirectional unwinding from theviral 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 duplexcircular 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. SV40Tantigeninteracts with Nbs1 to disrupt DNA replication control. Genes Dev. 18 (11), 1305–1316.

Yuzhakov, A., Kelman, Z., Hurwitz, J., O, Donnell, M., 1999. Multiple competitionreactions 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 proteasome-dependent destruction of Mre11-Rad50-Nbs1 subunits in simian virus 40-infectedprimate 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 notcellular-DNA replication. EMBO J. 15 (9), 2298–2305.

359Minireview