Vol. 11, No. 5

MOLECULAR AND CELLULAR BIOLOGY, May 1991, p. 2350-2361 0270-7306/91/052350-12$02.00/0 Copyright © 1991, American Society for Microbiology

Initiation of Simian Virus 40 DNA Synthesis In Vitro PETER A. BULLOCK,t* YEON SOO SEO, AND JERARD HURWITZ Graduate Program in Molecular Biology, Memorial Sloan-Kettering Cancer Institute, New York, New York 10021 Received 9 November 1990/Accepted 28 January 1991

Simian virus 40 (SV40) T antigen can efficiently initiate SV40 origin-dependent DNA synthesis in crude extracts of HeLa cells. Therefore, initiation of SV40 DNA synthesis can be analyzed in detail. We present evidence that antibodies which neutralize proliferating cell nuclear antigen (PCNA) inhibit but do not abolish pulse-labeling of nascent DNA. The lengths of DNA products formed after a 5-s pulse in the absence and presence of anti-PCNA serum averaged 150 and 34 nucleotides, respectively. The small DNAs formed in the presence of anti-PCNA serum underwent little or no increase in size during further incubation periods. The addition of PCNA to reaction mixtures inhibited with anti-PCNA serum largely reversed the inhibitory effect of the antiserum. The small nascent DNAs formed in the presence or absence of anti-PCNA serum products arose from the replication of lagging strands. These results suggest that a PCNA-dependent elongation reaction participates in the synthesis of lagging strands as well as leading strands. We also present evidence that in crude extracts of HeLa cells, DNA synthesis generally does not initiate within the core origin. Initiation of DNA synthesis outside of a genetically defined origin region has not been previously described in a eukaryotic replication system but appears to be a common feature of liitiation events in many prokaryotic organisms. Additional results presented indicate that in the absence of nucleoside triphosphates other than ATP, the preinitiation complex remains within or close to the SV40 origin. set of enzymes that includes DNA ligase, RNase H, and a 5'-to-3' exonuclease (23). Owing in part to techniques for producing large amounts of T-Ag (15, 40, 47), a greater understanding of the role played by SV40 T-Ag during initiation of replication has been obtained recently. The first critical function of T-Ag during initiation of DNA replication is that it must recognize and bind to the SV40 origin. The SV40 origin contains two major binding sites for T-Ag that are designated binding sites I and 11 (56; reviewed in references 3, 7, and 49). Binding site II is contained within a region of the origin, termed the core origin, which is necessary and sufficient for SV40 replication both in vitro and in vivo (references 2, 9, 13, 37, and 50 and references therein). Binding of T-Ag to site II is stimulated 10- to 15-fold in the presence of ATP (4, 12). Moreover, in the presence of ATP, T-Ag forms a complex nucleoprotein structure at the core origin (11) in which T-Ag is assembled as a double hexamer (32). The second critical function of T-Ag is its role in unwinding duplex DNA at the SV40 origin of replication (10, 16, 69). T-Ag initiates unwinding by melting an 8-bp region on the early side of the core origin; it concurrently changes the structure of an adenine-thymine tract on the late side (5, 41). The T-Ag-induced structural changes at the origin can be detected as a shift in the topological distribution of the covalently closed DNA molecules (45). Furthermore, when a topoisomerase capable of removing positive supercoils and an SSB are included in the reactions, T-Ag further unwinds SV40 origin-containing duplex DNAs (10, 16, 69). With circular duplex DNAs, the products of this reaction are a mixture of unwound circular DNAs termed form U. The ability of T-Ag to unwind SV40 origin-containing DNA is the result of an intrinsic helicase activity (48) that translocates in a 3'-to-5' direction (20, 64). Studies of the unwinding reaction conducted with purified proteins and plasmids containing point mutations in the SV40 core origin provided indirect evidence that unwinding of the SV40 origin is an essential initiation step during replication; those plasmids containing mutations that inacti-

With the exception of a single virus-encoded protein, the simian virus large tumor antigen (T-Ag), replication of simian virus 40 (SV40) DNA is dependent on host proteins from permissive simian or human cells (57). SV40 in vitro replication systems have been developed that require T-Ag, plasmids containing the SV40 origin, an ATP-regenerating system, and extracts prepared from either simian or human cells (30, 51, 66). Studies with the SV40 in vitro replication systems have enabled the identification of host proteins involved in SV40 and presumably chromosomal DNA replication (reviewed in references 7 and 49). As a result of these studies, the polymerase a (pol a)-primase complex was shown to be essential for SV40 DNA replication (30, 36) and to play a role in establishing the host range of SV40 DNA replication (36). Proliferating cell nuclear antigen (PCNA) and PCNA-dependent DNA pol 8 have been also demonstrated to be required for SV40 replication (26, 43, 70), especially for synthesis of leading strands (44). In addition, efficient elongation of newly synthesized DNA is known to require a factor termed activator 1. An inhibitor of DNA synthesis, poly(ADP-ribose) polymerase, copurifies with activator 1 through several isolation steps, but the two activities can be separated, and whether poly(ADP-ribose) polymerase plays a direct role in DNA synthesis is unknown (28, 29). A protein similar in activities and subunit structure to activator 1 has been isolated and termed RF-C (62). Topoisomerases have been shown to unlink DNA during progression of replication forks, and they segregate newly synthesized daughter molecules (23, 71). A multisubunit single-stranded DNA-binding protein (SSB), containing polypeptides of 70, 34, and 11 kDa, is also essential for SV40 DNA replication (19, 67, 68). Finally, it has been demonstrated that formation of covalently closed circles requires a * Corresponding author. t Present address: Department of Biochemistry, Tufts University, 136 Harrison Avenue, Boston, MA 02111.

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INITIATION OF SV40 DNA SYNTHESIS

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vated or markedly reduced their ability to replicate in vivo and in vitro were inactive in the unwinding assay (9). Direct evidence that unwinding is necessary for initiation of DNA synthesis was provided by pulse-chase experiments using crude extracts prepared from human HeLa cells, an ATPregenerating system, and T-Ag (6). After 10 min of incubation, the reaction mixtures contained a distinct unwound form of DNA that was termed form UR. On chloroquinecontaining agarose gels, form UR comigrated with a subset of the more heterogeneous population of unwound DNA molecules formed with purified proteins, form U. Pulse-chase experiments demonstrated that the first DNA species labeled comigrated with form UR and then matured into monomersize relaxed and supercoiled forms of DNA. When the products of the pulse-chase experiments were analyzed by restriction endonucleases, it was apparent that initiation occurred in the vicinity of the SV40 origin and then progressed outward in a bidirectional manner. These experiments established form UR as the earliest detectable substrate for SV40 DNA replication in vitro and demonstrated that with pulse-labeling techniques, SV40 DNA synthesis can be limited to very early events. However, in the previous study the location(s) within the SV40 origin region at which DNA synthesis initiated was not established. In this report, we present an analysis of the sites within the SV40 origin region that serve as templates for newly synthesized DNA at very early times. Moreover, to further characterize initiation events, we have examined the step during initiation of SV40 DNA synthesis in vitro at which PCNA is required for subsequent elongation events. MATERIALS AND METHODS Preparation of SV40 T-Ag, crude extracts, and antibodies against PCNA. SV40 T-Ag was immunoaffinity purified by using a baculovirus expression vector containing the T-Agencoding SV40 A gene (40). The preparation of HeLa cell extracts has been described previously (66). Anti-PCNA serum (AK) was generously provided by E. M. Tan of the Scripps Clinic and Research Foundation. Subcloning of pSV01AEP into M13mpl9. pSV01AEP (2,804 bp) was restricted with NcoI, HaeII, HincII, and PstI; restriction endonucleases were from New England BioLabs. Cleavage-generated fragments of 890, 668, 580, 370, and 2% bp are referred to as fragments A to E, respectively (see Fig. 2). The ends of these fragments were made blunt ended by treatment with T4 DNA polymerase and deoxynucleoside triphosphates (100 FxM, final concentration) (46), and fragments A to E were subsequently isolated from an agarose gel. Individual fragments were blunt end ligated with SmaIcleaved M13mpl9 that had been treated with calf intestinal phosphatase. Single-stranded DNA was isolated (46) from clones having the desired orientations, and the sequences of the inserts were confirmed by dideoxy DNA sequencing. Pulse-labeling. Reaction mixtures (120 ,ul) contained 7 mM MgCI2, 0.5 mM dithiothreitol, 4 mM ATP, 40 mM creatine phosphate (di-Tris salt; pH 7.7), 2.8 ,ug of creatine phosphokinase, 1.5 ,ug of supercoiled SV40 origin-containing pSV01AEP, 2 ,ug of T-Ag, and 60 ,u of HeLa cell extract (13.8 mg/ml). Reaction mixtures were preincubated for 45 min at 37°C in the absence of T-Ag and then further incubated for 15 min after the addition of T-Ag. As previously reported (6), the preincubation in the absence of T-Ag lowered the T-Ag-independent labeling of form II DNA (circular duplex DNA with at least one single-strand break). Reaction mixtures were pulse-labeled by the addition of a

2351

solution (7 pA) containing [a-32P]dCTP (final concentration, 1.0 ,uM; 253 cpm/fmol), dATP, dGTP, and dTTP (final concentration of each, 100 ,uM), and CTP, GTP, and UTP (final concentration of each, 200 pRM) for various times at 370C. Experiments designed to examine the role of PCNA on the initiation of DNA synthesis were conducted in a volume of 60 pul, rather than 120 pul, essentially as described above. However, 5 pI of anti-PCNA serum (AK) was included during the preincubation period. To demonstrate that inhibition by the AK serum was due to the selective inhibition of PCNA, purified PCNA (0.38 p.g in 25 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid [HEPES; pH 6.41-0.1 M KC1-0.5 mM EDTA-0.01% Nonidet P-40-10% glycerol20% sucrose, 1 mM dithrothreitol-0.1 mM phenylmethylsulfonyl fluoride; isolated as described previously [28] and supplied by T. Eki) was included in certain reaction mixtures during the preincubation period. Reactions were terminated by adding a mixture (12 RI; 6 pul for the small-volume anti-PCNA experiments) containing 15 mM EDTA, 4 pug of Escherichia coli tRNA, 0.3% sodium dodecyl sulfate (SDS), and 30 Rg of proteinase K. Aliquots (6.6 RI) were withdrawn to monitor incorporated label. All reaction mixtures were further incubated for 30 min at 37°C, diluted to 0.2 ml with 10 mM Tris-HCI (pH 7.8)-i mM EDTA, extracted with phenol-chloroform, adjusted to 2 M ammonium acetate, and precipitated with 2.5 volumes of ethanol. After centrifugation, the samples were reprecipitated with ammonium acetate (2 M) and ethanol and then washed with 80% (vol/vol) ethanol. After drying, the DNA pellets were resuspended in 150 pu1 of 10 mM Tris-HCl (pH 7.8)-i mM EDTA, and aliquots (containing 104 cpm) were applied to 1.8% alkaline agarose gels that were electrophoresed at 60 V for 15 h. The gels were either fixed in 8% (wt/vol) trichloroacetic acid and dried (Fig. 4) or transferred to Whatman DE81 paper (Fig. 7A) (46). Size markers used during alkaline gel electrophoresis were prepared as previously described (6). Additional aliquots (103 cpm) were applied to 10% polyacrylamide gels containing 8 M urea and electrophoresed at 1,000 V in lx TBE buffer (46). Where indicated, RNA primers were removed from nascent DNA molecules by incubating the samples in 0.3 N NaOH for 12 h. Alkali-treated samples were neutralized with HCI and ethanol precipitated after addition of 4 pI of 3.0 M sodium acetate. The pellets were washed with 70% ethanol and resuspended in 5 ,u of TE buffer. All samples were boiled for 2 min in 90% formamide before electrophoresis (46). Size markers included a [_y-32P]ATP-labeled oligo(dT)-22 ladder (Bethesda Research Laboratory) and a mixture containing a 5'-[^y-32P]ATP-labeled 1-kb ladder (Bethesda Research Laboratories) and 5'-[,y-32P]ATP-labeled oligo(dT)10 (Pharmacia-

LKB).

Pulse reactions conducted for brief

periods of time

re-

quired several reactions to attain sufficient incorporation for hybridization (approximately 105 cpm per hybridization). As a result, more substrate DNA was present in the pooled reactions from early time points than in the single reaction used to generate nascent DNA for the 5-min time point.

For this reason, upon pelleting of the samples, pSVO1AEP DNA was added to the resuspended pellets so that prior to hybridization, all samples contained equal amounts of pSVO1AEP DNA. Restriction analyses of DNAs labeled during a brief pulse. With the exception of the length of time T-Ag was preincubated in crude extracts prior to pulse-labeling, the method used to demonstrate that nascent DNAs were localized to

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MOL. CELL. BIOL.

BULLOCK ET AL.

the vicinity of the origin of replication was exactly as described in reference 6. Dot blot hybridization. DNA was affixed to Zeta Probe filters by using a Bio-Rad dot blot apparatus. To enable subsequent quantitation, the locations of the samples on the filters were marked with indelible ink. The filters were prehybridized in 50% formamide-5x SSC-150 ,ug of denatured salmon sperm DNA per ml-50 mM Tris-HCl (pH 7.5)-0.1% sodium pyrophosphate (tetrasodium)-5 mM EDTA-0.2% Ficoll (molecular weight 400,000), polyvinylpyrrolidone (molecular weight 40,000), 1% SDS, and 1% bovine serum albumin for 2 to 4 h at 42°C (hybridization buffer). The resuspended pulse reaction mixtures were heated to 95°C for 5 min and then added to the DNAcontaining Zeta Probe membranes in 7 ml of hybridization buffer. Following hybridization for 10 to 14 h at 42°C, the filters were washed twice in 2x SSC-0.1% SDS for 15 min at 65°C and then twice in 0.1% SSC-0.1% SDS for 30 min at 65°C. The filters were air dried and exposed to Kodak X-Omat AR film. After development of the films, relevant sections of the Zeta Probes were removed with a paper hole puncher and the extent of hybridization was determined by liquid scintillation counting. RESULTS

Nucleotide-dependent stabilization of the preinitiation complex at the SV40 origin. We previously reported that DNA synthesized during a 30-s pulse-labeling in HeLa cell crude extracts, after a 15-min preincubation in the presence of T-Ag, was localized to the SV40 origin region (6). This same result was obtained regardless of whether the preincubation in the presence of T-Ag was conducted for 15 min or 2 h (Fig. 1). After the various preincubation periods, reaction mixtures were pulse-labeled with ribonucleoside triphosphates, deoxynucleoside triphosphates, and [a-32P]dCTP for 15 s. To facilitate the formation of duplex DNA and the subsequent cleavage by restriction endonucleases, the pulselabeled products were chased with an excess of unlabeled dCTP for 30 min. The predominant localization of label in the origin-containing fragments suggests that in the absence of nucleotides other than ATP, the preinitiation complex was maintained in the vicinity of the origin. A densitometric trace of lane 3 in Fig. 1 indicated that 87% of the label was contained in origin-containing fragments B and C (see Fig. 2A for a map of pSVO1AEP). Furthermore, during long preincubation periods in the presence of T-Ag (up to 2 h), the mobility of form UR through chloroquine-containing agarose gels did not change (Sa). If T-Ag, in conjunction with HeLa SSB and a topoisomerase capable of removing positive supercoils, were to extensively unwind the DNA, a change in the mobility of form UR would be expected. The localization of label to the origin-containing fragments and the failure to detect a change in the mobility of form UR after long incubation periods suggest that in crude extracts, origin unwinding and initiation of DNA synthesis are coupled events. The possibility that these events are coupled was suggested previously (59). The significance of the experiments presented in Fig. 1 to the experiments described below is that once the preinitiation complex is formed, it does not appear to leave the vicinity of the origin until pulse-labeling. Most (approximately 90%) of the nascent DNAs formed during a brief pulse-labeling were from the origin region, and they are therefore not the products of aberrant synthesis events at distant single-stranded regions,

2 3 4

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FIG. 1. Initiation complex stabilization at the SV40 origin. Reaction mixtures were preincubated for the indicated times (15 min to 2 h) in the presence of HeLa crude extract, ATP, and an ATPregenerating system; reaction mixtures displayed in odd-numbered lanes were preincubated with T-Ag. Pulse-labeling was conducted for 15 s with ribonucleoside triphosphates, deoxynucleoside triphosphates, and [ot-32P]dCTP; after labeling, reactions were chased with an excess of unlabeled dCTP (Materials and Methods). Reactions were stopped, and the DNA was cleaved at the five restriction endonuclease sites shown in Fig. 2A. The sizes of the fragments resulting from cleavage of pSVOlAEP at the five sites are indicated at the left. Sequences from the SV40 origin containing the EcoRIl G fragment (Ori+) are located in the 668- and 580-bp fragments. Following restriction endonuclease digestion, the reaction mixtures were subjected to electrophoresis on a 5% polyacrylamide gel. + T-Ag present; -, T-Ag absent.

formed as the result of unwinding events during the preincubation period. Defining the regions in the vicinity of the SV40 origin that serve as templates for DNA synthesized during a brief pulse. A map of plasmid pSV01AEP is presented in Fig. 2A. The SV40 wild-type origin is contained in the 311-bp EcoRII G fragment depicted by the solid rectangle; the core origin is depicted by the smaller open rectangle. This plasmid was cleaved at the five indicated restriction endonuclease sites, and the resulting fragments were cloned into SinaI-cleaved M13mpl9, creating the pM13SVO1 series of clones (Materials and Methods). Clones containing both possible orientations of any given fragment were isolated; however, Fig. 2B presents maps of only those pM13SVO1 clones that contain DNA fragments complementary to lagging-strand DNAs. Clones containing fragments complementary to leadingstrand DNAs are in the opposite orientation. Sequences from the SV40 origin-containing EcoRII G fragment are present in the pM3SVOl C and B clones; however, the core origin is situated entirely within fragment B-containing clones (Fig. 2B). Since bidirectional DNA replication terminates in pSV01iEP fragment A, single-stranded DNA from fragment A cannot be used as a probe for either leading- or lagging-strand DNA synthesis. In Fig. 23, we have included the pMl3SVOl clone containing fragment A in an orientation that we are calling Al.

INITIATION OF SV40 DNA SYNTHESIS

VOL . 1 l, 1991

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FIG. 2. Establishment of the pM13SVO1 set of clones. (a) Restriction map of pSVO1AEP. Positions of the relevant restriction endonuclease sites are indicated. Symbols: _, the 311-bp SV40 origin-containing fragment cloned into this plasmid; the SV40 core origin. Numbers in parentheses represent SV40 nucleotides numbered according to Fiers (57); the other numbers represent nucleotide positions on plasmid pSVO1AEP. (b) Structures of clones. Plasmid pSVO1AEP fragments A to E (thicker lines) were cloned into the SmaI site of pMl3mpl9. Sequences from M13mpl9 are depicted by the thinner line. Clones having both possible orientations of a given fragment were isolated, but only those in an orientation complementary to nascent lagging-strand DNAs are shown. The number of nucleotides in each cloned fragment is shown above the thick lines. The names of the pM13SVO1 clones are indicated at the left. These clones are abbreviated B Lag to E Lag; those in the opposite orientation are abbreviated B Lead to E Lead. Included in the figure is the pMl3SVO1 clone containing fragment A in an orientation that we are calling Al; the clone having this fragment in the opposite orientation is referred to as A2. Blunt-end ligation of the pSVO1AEP fragments into the M13mpl9 SmaI site resulted in the loss of the terminal restriction sites in the fragments; this is symbolized by the absence of vertical bars at restriction endonuclease sites used to indicate fragment termini.

To determine the locations within the SV40 origin that

templates during initiation of DNA synthesis in crude extracts, nascent DNAs were formed by brief pulselabeling reactions. The labeled DNA products were then hybridized to single-stranded DNAs isolated from the pM13SV01 set of clones immobilized on Zeta Probe filters. In these experiments, the newly synthesized DNA was dissociated from the parental substrate DNA by heating; however, the nascent and substrate DNAs were not physically separated prior to hybridization. Results from a represerve as

2353

sentative series of experiments are shown in Fig. 3. After a 5-s pulse (Fig. 3A), the newly synthesized DNA hybridized preferentially to clone pM13SVO1 C Lag (abbreviated C Lag; other pM13SVO1 clones are abbreviated in a similar manner), a clone containing sequences complementary to nascent lagging-strand DNAs in the vicinity of the SV40 origin. There was also significant hybridization to B Lag, the second origin-containing clone having sequences complementary to nascent lagging-strand DNA. In contrast, there was little hybridization to C Lead or B Lead, clones containing sequences complementary to nascent leading-strand DNA derived from the origin region. To quantitate the extent of hybridization to these and the other pM13SV01 clones, DNA-containing regions of the Zeta Probe filters were removed and the amount of radioactivity was determined by liquid scintillation counting. The sum of the labeled DNA hybridized to the pM13SVO1 clones, minus the background hybridization to singlestranded m13 DNA, was calculated and used to establish the percentage of radioactivity hybridized to a given clone. The results of these analyses for the hybridization of nascent DNA to 1.0-,ug aliquots of the pM13SVO1 clones are presented to the right of the dot blots in Fig. 3. Similar results were obtained for the hybridization experiments carried out with the 0.1-,ig aliquots of the pM13SVO1 clones (data not shown). After a 5-s pulse, 40% of the probe hybridized to clone C Lag and 18.0o hybridized to clone B Lag. The percentages of hybridization to clones B Lead and C Lead were 6.5 and 5.5, respectively. Thus, of the total labeled DNA hybridized to the origin-containing pM13SVO1 clones (70% of the total), 83% hybridized to the lagging-strand probes. Clones containing fragments from flanking regions of pSVOlAEP showed less extensive hybridization to the nascent DNAs. There was also little hybridization of labeled DNA to single-stranded M13 DNA, the negative control, but extensive hybridization of newly synthesized DNA to denatured pSVOlAEP DNA, which served as a positive control. Finally, the preferential hybridization to C Lag relative to B Lag was a reproducible result (15 independent experiments). This finding indicates that upon pulse-labeling in vitro, DNA synthesis is initiated asymmetrically with respect to the SV40 origin. Similar results were obtained when products pulse-labeled with [a-32P]dTTP were used in place of dCTP (data not presented). Finally, we note that both in vivo and in vitro, topoisomerase I cleavage sites are located asymmetrically in the SV40 origin region predominantly on the strands that are the template for lagging-strand synthesis (42, 58). After a 1-min pulse (Fig. 3B), nascent DNA continued to hybridize preferentially to the origin-containing B and C clones. However, nascent DNAs complementary to the leading-strand B and C probes were more abundant, and the difference between the extent of hybridization to the laggingand leading-strand clones was reduced. Hybridization to B Lead and C Lead may result, in part, from elongation of the nascent DNA that was detected after a 5-s pulse. Thus, leading-strand synthesis may be primed by DNAs that, relative to the replication forks, are nascent lagging-strand DNAs (see Discussion). Finally, clones containing fragments from regions flanking the SV40 origin in pSVOlAEP continued to hybridize less extensively. After 5 min of pulse-labeling (Fig. 3C), the extent of hybridization to the individual pM13SV01 clones was approximately 10%. Since there were 10 pM13SV01 clones,

approximately 10%o hybridization to any given pM13SV01 clone represented random hybridization. However, as one

2354

MOL. CELL. BIOL.

BULLOCK ET AL.

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DSVO'\ EP @0 set of clones with nascent DNA. Nascent DNAs formed after 5 s, 1 min, or 5 min of DNA pM13SVO1 of the 3. Dot blot hybridizations FIG. synthesis in vitro were hybridized to the pM13SVO1 series of clones immobilized on Zeta Probe membranes (Fig. 3A to C, respectively). For each clone, aliquots containing 0.1 and 1.0 ,ug of DNA were immobilized. Hybridization controls included M13mpl9 single-stranded DNA (M13) and denatured pSVO1AEP DNA. Bar graphs showing the percentage of the total counts hybridized to the 1-,ug aliquots of the pM13SVO1 clones are presented at the right. Leading and Lagging refer to the nascent DNA. For the 5-s, 1-min, and 5-min time points, the sums of the radioactivity hybridized to the 1-,ig aliquots of the pM13SVO1 clones (100% hybridization) were 1,041, 1,121, and 1,899 cpm, respectively.

would predict, slightly less hybridization was detected with the pM13SVO1 clones containing smaller inserts. Moreover, when nick-translated pSVO01AEP DNA was used to probe identical filters, the hybridization pattern was similar to the one described in Fig. 3C (data not shown). Size distribution of nascent DNAs. The length of the DNA synthesized in the pulse-labeling reactions was determined by alkaline gel electrophoresis (Fig. 4). The size distribution of nascent DNA formed after 5 s of synthesis is shown in lane 3. This lane contained an aliquot (104 cpm) of the sample used in the hybridization experiments shown in Fig. 3A. The size distribution of newly synthesized DNA ranged between -40 and 322 bp. The peak of nascent DNA migrated slightly below the 185- and 174-bp size markers. An aliquot (104 cpm) of the sample used in the hybridization experiment presented in Fig. 3B, containing nascent DNAs formed after 1 min of DNA synthesis, is shown in Fig. 4, lane 4. Relative to the population of nascent DNA formed after a 5-s pulse, this population consisted of longer nascent DNA. An aliquot (104 cpm) of the sample used in the hybridization experiment shown in Fig. 3C, formed after 5 min of DNA synthesis, is presented in Fig. 4, lane 5. This distribution included DNA

that comigrated with the 641-bp size marker and larger DNA that comigrated with single-stranded circular forms of pSV01AEP. The DNA formed after a 5-min pulse in the absence of T-Ag consisted of single-stranded circular and single-stranded linear forms of pSV01AEP DNA (lane 7). The products of the entire reaction were loaded in this lane to show the positions of the two forms of DNA produced by non-T-Ag (as well as non-PCNA [see below])-dependent DNA synthesis. Most likely, these labeled products were derived from spurious end labeling of randomly nicked DNA (single-stranded linear) followed by partial ligation (singlestrand circular). After a brief pulse, labeled DNA could be detected in non-origin-containing pSVO1AEP fragments (Fig. 1 and 3). When analyzed by restriction endonuclease cleavage of pulse-chase reactions (Fig. 1), approximately 13% of the label was in the non-origin-containing fragments; 30% of the label was in the non-origin-containing fragments when assayed by hybridization (Fig. 3A). Thus, our experiments raise the possibility that a small percentage of the initiation events in our reactions may be occurring outside of the origin region. Others have reported that at a low frequency,

VOL. 11, 1991

2355

INITIATION OF SV40 DNA SYNTHESIS

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FIG. 4. Determination of the sizes of the DNAs synthesized during pulse-labeling. Aliquots from reaction mixtures pulse-labeled for 5 s, 1 min, and 5 min (104 cpm for each time point) were removed, loaded, and electrophoresed on a 1.8% alkaline agarose gel. Lanes 1 and 7 contained the entire labeled products formed after 5 s or 5 min of pulse-labeling in the absence of T-Ag. The positions of singlestranded linear (ssl) and single-stranded circular (ssC) DNAs are indicated. The sizes (in base pairs) of marker (M) DNA, prepared as previously described (6), are also indicated. + and - indicate presence and absence, respectively, of T-Ag.

initiation events occur outside of the SV40 origin in vivo (31). Regarding the higher percentage of label in the nonorigin-containing fragments when assayed by hybridization, the size distribution of nascent DNAs (Fig. 4) suggests that hybridization to the 3' ends of the longer members of the nascent DNA population may account, in part, for some of the hybridization (Fig. 3A). However, fragment A is over 800 nucleotides (nt) away from the SV40 origin, and the size distribution of the nascent DNAs suggests that newly synthesized DNAs did not contain sequences complementary to fragment A. In view of this observation, and the lower percentage of label found in the non-origin-containing fragments presented in Fig. 1 (approximately 13%), it is likely that hybridization to the pM13SVO1 clones included a low level of background hybridization. We found that the level of hybridization to the non-origin-containing fragments was reduced, but not eliminated, when nascent DNAs formed by a brief pulse were separated from substrate pSVO1AEP DNA by alkaline sucrose centrifugation prior to hybridization (5a). Influence of antibodies that neutralie PCNA and their effects on pulse-labeling of nascent DNA. The results presented above indicate that DNA synthesized after a short pulse-labeling period arises from the replication of lagging strands. Since DNA pol a is complexed to DNA primase, it is likely that the initiation of DNA synthesis occurs by the coupled formation of small RNA primers by DNA primase which are immediately elongated by DNA pOl a. In keeping with this proposal, low levels (10 ,uM) of N2-[p(n-butyl) phenyl] deoxyguanosine 5-triphosphate (BuPdGTP), a selective inhibitor of pol a at low concentrations, blocked the labeling of form UR after a 20-s pulse (6). PCNA and Al (RF-C) are essential for the pol B-catalyzed

FIG. 5. Effects of anti-PCNA serum on the sizes of the products formed during pulse-labelings. Reaction mixtures (60 p.l) were pulse-labeled for 5 s, 45 s, or 5 min and subsequently electrophoresed on a 1.8% alkaline agarose gel. The products formed after 5 s, 45 s, and 5 min of pulse-labeling under standard conditions are shown in lanes 2, 7, and 12, respectively; the products formed after identical pulse-labelings but in the presence of 5 ,ul of anti-PCNA serum (AK) are shown in lanes 3, 8, and 13. To demonstrate that the decrease in size was due to selective inhibition of PCNA by the anti-PCNA serum, purified PCNA (0.38 ,ug) was added to the reaction mixtures displayed in lanes 4, 9, and 14. The effects of the same amount of purified PCNA on pulse reactions lacking antiPCNA serum were also determined, and the products of these reactions are displayed in lanes 5, 10, and 15. Lanes 1, 6, and 11 contain the products formed after pulse-labeling for 5 s, 45 s, and 5 min in the absence of T-Ag. The positions of single-stranded linear (ssl) and single-stranded circular (ssc) DNAs are indicated. Lane 16 contains size markers from a kinase-treated 1-kb DNA ladder (Bethesda Research Laboratories); the fragment sizes (in base pairs) are indicated at the right. + and - indicate the presence and absence, respectively, of the indicated components.

elongation of primed DNA templates but appear to play no role in the initiation reactions catalyzed by the pol a-primase complex (26, 49). Furthermore, sera from patients with lupus erythematosus contain antibodies which neutralize PCNA (34, 53), and SV40 replication reactions in crude extracts of HeLa cells were inhibited (90%) by such sera; highly purified HeLa PCNA reversed this inhibition (26). These findings prompted us to examine the effects of such antisera on the pulse-labeled synthesis of DNA (Fig. 5). Levels of anti-PCNA serum were used which inhibited the standard 60-min replication assay 80 to 90% and were quantitatively reversed by 0.38 ,ug of PCNA (data not presented). Crude extracts, in the presence of this level of serum and T-Ag, were preincubated for 15 min and then pulse-labeled for either 5 s, 45 s, or 5 min; preincubations were also carried out with crude extracts with serum plus added PCNA at concentrations which reversed the inhibition (Materials and Methods). At all time points, the anti-PCNA serum markedly inhibited, but did not eliminate, the amount of DNA synthesized (Fig. 5, lanes 3, 8, and 13). The addition of PCNA to neutralize the anti-PCNA antibodies reversed the marked inhibition of the pulse-labeling reaction (lanes 4, 9, and 14). After a 5-s pulse, the inhibition of the pulselabeling reaction was only partially reversed by purified

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FIG. 6. (A) Dot blot hybridization of the pM13SVO 1 set of clones with nascent DNA formed during pulse-labeling in thie presence of anti-PCNA serum. The small nascent DNA products fformed after 5 min of pulse-labeling in the presence of anti-PCNA seruum (the entire reaction mixture, average size approximately 75 nt; sere Fig. 5, lane 13) were hybridized to the pM13SVO1 series of clone: s immobilized on Zeta Probe membranes. Aliquots containing 0.1;and 1.0 of each clone were immobilized to the filters. Hybridiz; included M13mpl9 single-stranded DNA and denatur ed DNA. (B) Bar graph showing the percentage of the total counts hybridized to the 1-,ug aliquots of the pM13SVO1 clc)nes. Leading and Lagging refer to the nascent DNA. The sum of the radioactivity hybridized to the 1-,ug aliquots of the pM13SVO1 clones (100% hybridization) was 901 cpm.

cng

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PCNA (lane 4). However, 5-s pulse reactions devoid of anti-PCNA serum were also partially inhibited by purified PCNA (lane 5), and purified PCNA also had a slight inhibitory effect on the 45-s and 5-min pulse reactions which were not treated with the anti-PCNA serum (lanes 10 and 15). Additional studies have shown that the PCNA buffer (Materials and Methods) slightly inhibited pulse-labeling of DNA (unpublished data). These studies demonstrated that antiPCNA serum limited the formation of nascent DNA to a very small size during initiation of synthesis and that the inhibition was, in general, reversed by the addition of purified PCNA. The small DNA products formed in the presence of the anti-PCNA serum after pulse-labeling for 5 min were analyzed by dot- blot hybridization. Hybridization of the entire reaction products occurred preferentially to pM13SVO1 clones C Lag and B Lag (Fig. 6). A comparison of the

hybridization patterns in Fig. 6 and Fig. 3A indicated that the nascent DNA present 5 min after initiation of synthesis in the presence of anti-PCNA serum was similar to the products formed after a 5-s pulse in the absence of anti-PCNA serum. Nascent DNA products formed after 1 min of pulselabeling in the presence or absence of anti-PCNA serum were subjected to urea-polyacrylamide gel electrophoresis (Fig. 7B). To determine the overall size distribution of the nascent DNA, aliquots from these reaction mixtures were also subjected to alkaline gel electrophoresis (Fig. 7A). The products formed in the presence of anti-PCNA serum included a small species (centered at approximately 34 nt; Fig. 7B, arrowhead) and additional minor bands (Fig. 7B, lane 5). Nascent DNA formed in the absence of anti-PCNA serum also contained a species -34 nt long; however, relative to reactions in which anti-PCNA serum was present, it was less prevalent (Fig. 7B, lane 2). Regarding the higher-molecularweight species present in lane 2 but absent in lane 5, after a 1-min pulse, nascent DNA formed in the absence of antiPCNA serum had an average size of -150 nt (Fig. 7A, lane 2), while nascent DNA formed in the presence of anti-PCNA serum did not extend much beyond 75 nt (Fig. 7A, lane 3). Thus, the high-molecular-weight species in Fig. 7B, lane 2, were PCNA-dependent elongation products. Aliquots of the nascent DNA products were also hydrolyzed in alkali (0.3 NaOH) for 12 h prior to urea-polyacrylamide gel electrophoresis (Fig. 7B, lanes 1 and 6). Alkaline hydrolysis of the products formed in the presence or absence of anti-PCNA serum reduced the length of the -34-nt species to -24 nt (arrow in Fig. 7B). The decrease in length due to NaOH treatment suggests that the -34-nt DNA chains were covalently linked to oligoribonucleotides 10 nt long. Nethanel et al. (39) studied SV40 DNA replication in isolated nuclei. In the presence of aphidicolin, they observed precursors to Okazaki-size fragments that they termed primer-DNA. Primer-DNA chains, approximately 40 nt in length, were derived from lagging-strand templates, and many molecules contained RNA chains approximately 10 nt long which were covalently linked to the DNA chains. In view of similarities between the results reported here and those of Nethanel et al. (39), we will refer to the -34-nt DNA fragments as primer-DNA. However, we define the term primer-DNA to include only the pol a-primase-dependent,

PCNA-independent initiation products. DISCUSSION The pulse-chase experiments presented in this study and those previously described (6) were carried out with crude extracts rather than with purified proteins. One reason for using crude extracts is that origin unwinding and initiation events appear to be coupled during SV40 replication, and we are uncertain of the requirements for coupling. Moreover, we have repeatedly observed that the pulse-labeling of SV40 origin-containing DNA is 20- to 40-fold more efficient with crude extracts of HeLa cells than with replication systems reconstituted with purified proteins (5a). The purified systems include the monopolymerase system (T-Ag, human SSB, topoisomerase I, and pol a-primase) and the dipolymerase system (PCNA, PCNA-dependent DNA pol 8, activator 1, and the components of the monopolymerase system) (23, 26, 67). The purified systems were reconstituted with proteins whose enzymatic activities were equal to or greater than the same activities in the crude extracts. Thus, a factor(s) required for efficient initiation, that may play a role

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in coupling origin unwinding and DNA synthesis events, appears to be missing from the purified systems. Indeed, when similar pulse experiments were carried out with the monopolymerase system, pulse-labeling (for 5 min) resulted in a more random initiation of chains (unpublished results). When DNA synthesis was carried out without preincubation in the monopolymerase system, sequences in the vicinity of the origin were labeled first, followed by the bidirectional labeling of regions further away from the origin. These results suggest that unlike the crude system, extensive DNA unwinding during preincubation in the purified system generated multiple sites at which the pol a-primase complex initiated synthesis. Previous analysis of initiation of SV40 DNA replication in vivo indicated that DNA synthesis starts in the vicinity of the BglI site within the SV40 origin (8). Additional in vivo studies have, in general, supported these results. For example, the 5' ends of nascent DNAs formed in vivo after a 30-min labeling period have been mapped (22). It was reported that the 5' ends of the newly synthesized DNA were derived from many sites in the vicinity of the SV40 origin. Some of these sites were within the core origin region, but only on the strand that served as the template for early mRNA. The 5' ends of nascent DNA in the opposite orientation mapped exclusively outside of the core origin on the early-gene side. On the basis of these studies, a model was proposed suggesting that a single initiation event first occurs within the SV40 core origin and then is elongated into DNA having the same polarity as early mRNA. A prediction of this model is that newly initiated DNAs should hybridize preferentially to the pM13SV01 clone B Lead. However, our studies indicate that nascent DNA formed after 5 s of DNA synthesis in vitro hybridized preferentially to clone C Lag and, to a lesser extent, to clone B Lag. Extensive hybridization to clone B Lead and to clone C Lead were detected only at time points after hybridization to clone C Lag and B Lag had already been detected. These data indicate that under the in vitro conditions used for pulse-labeling, laggingstrand synthesis preceded leading-strand synthesis. An additional feature of SV40 initiation events in vitro is that the replication machinery does not appear to initiate within the core origin. The 64-bp SV40 core origin extends between SV40 nt 31 to 5211, a region contained entirely within the B fragment of pSVO1AEP (Fig. 2 and 8). The core origin contains all of the sequence elements required for initiation of viral DNA replication both in vitro and in vivo (for reviews, see references 3, 7, and 49). Functional do-

-15

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+

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FIG. 7. Analyses of the products formed during a 1-min pulse in the presence or absence of anti-PCNA serum. (A) Nascent DNAs were formed during a 1-min pulse in either the presence (+) or absence (-) of anti-PCNA serum. The overall size distributions of the nascent DNAs were determined by alkaline gel electrophoresis of aliquots (containing 1,000 cpm) from the initial reactions. Lane 2 was loaded with nascent DNA formed in the absence of anti-PCNA serum. Nascent DNA formed after a 1-min pulse in the presence of

anti-PCNA serum is presented in lane 3. The products formed after a 1-min pulse in the absence of both T-Ag and anti-PCNA serum are shown in lane 1. The positions of single-stranded linear (ssl) and single-stranded circular (ssc) DNAs are indicated. The size markers in lane 4 are the same as those described in the legend to Fig. 5; the fragment lengths (in base pairs) are indicated at the right. (B) Aliquots containing 1,000 cpm from the reactions shown in panel A were run on a 10%o polyacrylamide-urea gel. The products formed in the absence (-) of anti-PCNA serum are shown in lanes 1 and 2; those formed in the presence (+) of anti-PCNA serum are shown in lanes 5 and 6. The samples displayed in lane 1 and lane 6 were treated with alkali prior to electrophoresis. The arrowhead indicates the -34-nt primer-DNA; the arrow indicates the position of the primer-DNA after treatment with alkali. Marker DNAs include an oligo(dT)-22 ladder (lane 3) and an oligo(dT)1O fragment mixed with a 1-kb DNA ladder (lane 4); the fragment sizes are as indicated (in base pairs).

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NcoI 2608 (37)

FIG. 8. Model of the initiation of SV40 DNA synthesis after pulse-labeling in vitro. The 311-bp SV40 origin-containing EcoRII fragment, extending between SV40 DNA nt 160 to 5092, is depicted by the solid rectangles; the smaller open rectangles depict the SV40 core origin. Numbers within parentheses represent SV40 nucleotides numbered according to Fiers (57). The vertical dashed line indicates the position of the NcoI restriction site. DNAs in the vicinity of the SV40 origin that can serve as templates during initiation of DNA synthesis are contained in the four pM13SVO1 clones, C Lead, C Lag, B Lead, and B Lag. The small arrows indicate the PCNA-independent, RNA-containing, -34-nt primerDNA. The longer arrows depict the PCNA-dependent extension of the primer-DNA. The positions of the arrows indicate the regions where DNA synthesis initiates after pulse-labeling in vitro. Preferential hybridization to C Lag suggests that early DNA synthesis events are more frequent than late synthesis events; this is indicated by the use of arrows of different thickness. Early and Late refer to the early and late SV40 genes (57). The model presented summarizes results obtained from more than 15 separate experiments.

mains within the core origin have been reported to be interaction sites for protein components of the initiation complex (13). Nevertheless, our studies indicate that in extracts prepared from HeLa cells, DNA synthesis initiates on either strand 3' to the core origin. Were DNA synthesis events to initiate in the core origin, nascent DNAs formed after brief labeling periods would hybridize preferentially to clones B Lead and C Lead (Fig. 8). Moreover, the size distribution of the nascent DNA (Fig. 4, lane 3) suggests that in vitro, some initiation events may occur entirely outside of the 322-bp SV40 origin-containing insert. To illustrate this point, if nascent DNA in the .200-nt size class were synthesized from sequences on the lower template, centered at the NcoI site, equal hybridization to the C Lag and B Lead clones would be expected (Fig. 8). However, nascent DNA preferentially hybridized to clone C Lag (Fig. 3A). Since there are only 123 bp of SV40 sequences between the NcoI site at nt 37 (the terminus of fragment C) and the EcoRII site at nt 160, the 5' ends of nascent DNA in the approximately 200 nt and larger size class map to vector sequences beyond the EcoRII site at nt 160. By similar reasoning, the 5' ends of some of the nascent DNAs initiating in B Lag may be encoded by vector sequences beyond the SV40 EcoRII at site nt 5092. We are currently mapping the 5' termini of the nascent DNA formed after a 5-s pulse-labeling. Studies of the sites at which DNA synthesis initiates in prokaryotic systems provide many precedents for initiation events occurring near, but outside, genetically defined origins. For example, the labeling pattern in vitro of plasmids containing the origin of replication of the E. coli chromooriC, suggests that synthesis initiates in a region 67 to 122 bp outside of the oriC sequence (52). Similar patterns of initiation were obtained when oriC-containing plasmids with different sequences at the actual initiation sites were used. It was concluded that the oriC sequence contained all of the information required to direct bidirectional replication at an adjacent region. Analogous results have been obtained from some,

studies of bacteriophage T7 (54), 4X (1), X(72), and Adv (60) genomes as well as analyses of ColEl (24, 33) and oriC replication in vivo (25). In light of these reports and the experiments presented here, it is possible that eukaryotic and prokaryotic initiation mechanisms are evolutionarily related. Reports indicating that several of the enzymatic activities required for SV40 replication have prokaryotic analogs support this hypothesis. For example, it was recently suggested that RF-C and PCNA have functions analogous to those of the proteins encoded by the bacteriophage T4 genes 44/62 and 45 (63). They also have functions analogous to the DNA pol III accessory proteins -y and 8 (T) and the dnaN gene product (27). The reason(s) for the differences between the reported sites of initiation of SV40 DNA synthesis in vitro and in vivo is unclear. One possibility is that the in vitro replication systems do not fully mimic initiation events in vivo. For example, to allow a functional preinitiation complex to form, the in vitro reactions were preincubated in the presence of T-Ag for 15 min prior to pulse-labeling. This preincubation period is slightly longer than the time required, approximately 10 min, to establish a functional preinitiation complex (18, 65). The extent to which the preincubation mimics conditions in vivo is not known. The experiments presented in Fig. 1 indicate that in the absence of deoxynucleoside triphosphates, the preinitiation complex is stabilized in the vicinity of the SV40 origin. However, our finding that initiation events occur in crude extracts distal to the core origin may be due to more unwinding than actually occurs in vivo. An alternative possibility is that since the in vitro mapping studies were conducted with nascent DNA formed after as little as 5 s of synthesis, whereas the in vivo studies were conducted with DNA formed after 30 min of synthesis, the in vitro results are a more accurate reflection of initiation events. It should be noted that although the in vivo-derived model for SV40 DNA replication (22) suggests that DNA synthesis is begun by a single initiation event within the core origin, several initiation events outside of core were actually observed. We also note that studies of initiation of SV40 DNA synthesis in vivo actually provide additional evidence for initiation events occurring outside of the core origin region. For example, consider the mapping studies presented by Hay and DePamphilis (21). The majority of the nascent DNA having the same sense as early mRNA migrated on an acrylamide gel with a mobility that suggests that it is 100 to 200 bp larger than the fragments terminating in the vicinity of the BglI site. On a lower-percentage acrylamide gel, initiation sites may have been mapped to regions similar to what we have observed in vitro. Initiation of synthesis with DNA that, relative to the replication forks, is lagging-strand DNA is consistent with what is known of the enzymology of initiation of SV40 DNA synthesis. For example, it has been reported that PCNA is required for synthesis of leading, but not lagging, strands in replication reactions conducted for 30 min using partially purified fractions (44). A similar conclusion was drawn based on hybridization studies using nascent DNA formed with purified proteins after 60 min of DNA synthesis (61). That PCNA and PCNA-dependent pol 8 are involved in elongation but not initiation events is also suggested by the observation that pol 8 does not have an associated priming activity (55). The experiments presented in Fig. 5 and 6 demonstrate directly that PCNA is not required to initiate lagging-strand DNA synthesis. Pulse-labeling of the SV40 replication reactions in the presence of anti-PCNA serum markedly re-

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duced, but did not eliminate, synthesis of DNA. The primerDNA chains formed in the presence of anti-PCNA serum specifically hybridized to pM13SVO1 clones B Lag and C Lag. We previously noted that pulse-labeling was eliminated by BuPdGTP, a selective inhibitor of pol a at low concentrations. These observations suggest that the pol a-primase complex synthesizes primer-DNA with use of lagging-strand templates. Primer-DNAs, which are observed in reactions even in the absence of anti-PCNA serum, are likely to serve as primers for subsequent PCNA-dependent pol 8, or perhaps PCNA-dependent pol £ (35 and references therein), elongation events. The recent observation that pol 8 isolated from HeLa cells extended DNA primers efficiently and RNA primers poorly is consistent with this possibility (26). The small size of the primer DNA suggests that the switch from the pol a-catalyzed initiation reaction to a PCNA-dependent elongation event occurs quite soon after initiation of synthesis. Finally, it is noted that Eliasson and Reichard (17), who studied replication of polyomavirus DNA in isolated nuclei, reported the accumulation of an -30-nt-long nascent DNA linked to RNA after initiation of DNA synthesis. It is suggested that they were observing pol a-primase-dependent primer-DNA formation. Anti-PCNA serum prevented the appearance of Okazakisize DNA fragments (-200 nt) during either 45-s or 5-min pulse-labeling (Fig. 5). This observation suggests that PCNA-dependent elongation events not only include formation of leading strands but may also include maturation of pol a-primase-dependent primer-DNA into Okazaki fragments on lagging strands. Recent studies of SV40 synthesis events in isolated nuclei also indicate that two polymerases may be involved in the maturation of Okazaki fragments (38). Furthermore, we previously showed that poly(ADP-ribose) polymerase can bind to 3' ends of DNA formed by pol a-primase and block further elongation events. This block was efficiently reversed by the addition of Al, PCNA, and pol 8 (29). Collectively, these observations suggest that maturation of Okazaki fragments requires two distinct DNA polymerases. However, in light of recent report that three DNA polymerases are essential in yeast cells (35), we are uncertain about the roles played by the PCNA-dependent polymerases during elongation events. It should be emphasized that primer-DNA formed after a 5-min pulse in the presence of anti-PCNA antibodies hybridized preferentially to B Lag and C Lag. This result differs from those of Prelich and Stillman (44), who reported that removal of PCNA arrested leading-strand DNA synthesis, though lagging-strand synthesis proceeded normally. Thus, in contrast to the studies of Prelich and Stillman, the failure to detect extensive hybridization to the other pM13SVO1 Lag clones indicates that in our system, leading- and laggingstrand synthesis events are coupled even in the absence of PCNA. The coupling of leading- and lagging-strand synthesis events under standard conditions in the pulse (Fig. 4) and pulse-chase (6) reactions is suggested by the failure to detect Okazaki-size fragments at points after elongation events are initiated. Our studies suggest a general mechanism of initiation of SV40 DNA synthesis in vitro that is presented in Fig. 8. The small arrows represent pol a-primase-dependent primer DNAs, while the longer arrows represent PCNA-dependent elongation of the primer-DNA. We are currently uncertain whether the initial primer-DNAs are unique or consist of a more heterogeneous population of molecules. The arrows are positioned in the vicinity of the SV40 origin where DNA synthesis initiates upon pulse-labeling in vitro. Our experi-

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ments indicate that on either strand, most initiation events do not occur within the core origin and that initiation events outside of the 311-bp EcoRII fragment may occur. Preferential initiation of DNA synthesis on the lower strand is symbolized by the thicker arrow. It should be noted that asymmetric initiation events are a feature of many prokaryotic replication systems (24, 25, 60) and have also been suggested for SV40 initiation events in vivo (14). Subsequent to the formation of primer-DNAs and the elongation events depicted in Fig. 8, pol a-primase is free to move, possibly in association with T-Ag, to form additional primer-DNAs, a likely prerequisite for Okazaki fragment formation. Our experiments and those of Nethanel et al. (38) suggest that maturation of primer-DNA into Okazaki fragments requires a PCNA-dependent elongation event. Further studies are now in progress to test this model. ACKNOWLEDGMENTS We thank N. Belgado and B. Phillips for technical assistance. We also gratefully acknowledge M. Lusky, J. Borowiec, F. Dean, K. Marians, and P. Traktman for helpful discussions. This work was supported by National Institutes of Health grant GM-34559-06. P.A.B. was supported by a fellowship from the American Cancer Society. REFERENCES 1. Arai, K., and A. Kornberg. 1981. Unique primed start of phage 4X174 DNA replication and mobility of the primosome in a direction opposite chain synthesis. Proc. Natl. Acad. Sci. USA 78:69-73. 2. Bergsma, D. J., D. M. Olive, S. W. Hartzefl, and K. N. Subrnman. 1982. Territorial limits and functional anatomy of the simian virus 40 replication origin. Proc. Natl. Acad. Sci. USA 79:381-385. 3. Borowiec, J. A., F. B. Dean, P. A. Bullock, and J. Hurwitz. 1990. Binding and unwinding-how T antigen engages the SV40 origin of DNA replication. Cell 60:181-184. 4. Borowiec, J. A., and J. Hurwitz. 1988. ATP stimulates the binding of simian virus 40 (SV40) large tumor antigen to the SV40 origin of replication. Proc. Natl. Acad. Sci. USA 85:6468. 5. Borowiec, J. A., and J. Hurwitz. 1988. Localized melting and structural changes in the SV40 origin of replication induced by T-antigen. EMBO J. 7:3149-3158. 5a.Bullock, P. A., and J. Hurwitz. Unpublished data. 6. Buflock, P. A., Y. S. Seo, and J. Hurwitz. 1989. Initiation of simian virus 40 DNA replication in vitro: pulse-chase experiments identify the first labeled species as topologically unwound. Proc. Natl. Acad. Sci. USA 86:3944-3948. 7. Chaflberg, M. D., and T. J. Kefly. 1989. Animal virus DNA replication. Annu. Rev. Biochem. 58:671-717. 8. Danna, K. J., and D. Nathans. 1972. Bidirectional replication of simian virus 40 DNA. Proc. Natl. Acad. Sci. USA 69:30973100. 9. Dean, F. B., J. A. Borowiec, Y. Ishimi, S. Deb, P. Tegtmeyer, and J. Hurwitz. 1987. Simian virus 40 large tumor antigen requires three core replication origin domains for DNA unwinding and replication in vitro. Proc. Natl. Acad. Sci. USA 84: 8267-8271. 10. Dean, F. B., P. Bullock, Y. Murakami, C. R. Wobbe, L. Weissbach, and J. Hurwitz. 1987. Simian virus 40 (SV40) DNA replication: SV40 large T antigen unwinds DNA containing the SV40 origin of replication. Proc. Natl. Acad. Sci. USA 84:1620. 11. Dean, F. B., M. Dodson, H. Echols, and J. Hurwitz. 1987. ATP-dependent formation of a specialized nucleoprotein structure by simian virus 40 (SV40) large tumor antigen at the SV40 replication origin. Proc. Natl. Acad. Sci. USA 84:8981-8985. 12. Deb, S. P., and P. Tegtmeyer. 1987. ATP enhances the binding of simian virus 40 large T antigen to the origin of replication. J.

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Initiation of simian virus 40 DNA synthesis in vitro.

Simian virus 40 (SV40) T antigen can efficiently initiate SV40 origin-dependent DNA synthesis in crude extracts of HeLa cells. Therefore, initiation o...
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