Replication Factors Required for SV40 DNA Replication in Vitro

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Replication Factors Required for SV40 DNA Replication in Vitro. 11. SWITCHING OF DNA POLYMERASE a AND 6 DURING INITIATION OF LEADING AND ...
Val. 266. No 3. Issue of January 25. pp. 1961-1968.1991 Printed in U S.A.

OF BIOLOGICALCHEMISTRY THEJOURNAL 0 1991 by The American Society for Biochemistry and Molecular Biology, Inc.

Replication Factors Required forSV40 DNA Replication in Vitro 11. SWITCHING OF DNA POLYMERASE a AND 6 DURING INITIATION OF LEADING AND LAGGING STRAND SYNTHESIS* (Received for publication, July 26, 1990)

Toshiki Tsurimoto and Bruce Stillman From the Cold Spring Harbor Laboratory, Cold Spring Harbor, New York 1 1 724

kDa), and 11,000 (11kDa) (Wobbe et al., 1987; Fairman and Stillman, 1988; Wold and Kelly, 1988), with the 70-kDa subunit functioning as a single strand-specific DNA binding protein (Brill and Stillman, 1989; Woldet al., 1989; Kenny et al., 1990). In the absence of DNA synthesis, TAg, topoisomerase, and RF-A cooperate to extensively unwind plasmid DNAs containing the SV40 origin of replication (Dean et al., 1987; Dodson et al., 1987; Wold et al., 1987; Borowiec and Hurwitz, 1988; Goetz et al., 1988; Wiekowski et al., 1988; Roberts, 1989) but in the presence of the full complement of replication factors, origin unwinding and initiation of DNA replication are coupled (Tsurimoto et al., 1989). The initiation of actual DNA synthesis at the replication origin first involves the interaction of DNA polymerase aprimase (pol a ) with the “unwound complex,” a complex of TAg and RF-A stably boundat theorigin of DNA replication (Lee et al., 1989; Tsurimoto et al., 1989; Borowiec et al., 1990; Tsurimoto et al., 1990). Initiation of DNA synthesis most likely involves the formation of aprimer by the primase activity associated with DNA polymerase a and synthesis of an Okazaki fragment at the origin. Pol OL then continues to synthesize Okazaki fragments exclusivelyon the lagging , although under some strand template (Tsurirnotoet ~ l .1990), abnormal conditions, pol OL can copy both leading and lagging strand templates (Ishimi et al., 1988). For complete DNA replication of the leading strand DNA template, three additional replication factors have been idenThe identification and characterization of the cellular pro- tified. Replication factor C (RF-C) is a multisubunit enzyme teins required for simian virus 40 (SV40) DNA replication in consisting of polypeptides with relative molecular masses of vitro has opened the way for detailed studies on the mecha- 140,000, 41,000, and 37,000 (140 kDa, 41 kDa, and 37 kDa, nism of DNA replication (reviewed in Challberg and Kelly, respectively) (Tsurimoto andStillman, 1989a). RF-C binds in 1989; Stillman, 1989; Borowiec et aL, 1990). SV40 DNA rep- a structurally specific manner to a primer-template junction lication has been reconstituted in vitro with purified sV40 and also has a DNA-dependent ATPase activity (Tsurimoto large tumor antigen (TAg)’ and seven essential cellular pro- and Stillman, 1990,1991). The RF-C.DNA-dependent ATPteins (Tsurimoto et al., 1990). Based upon mechanistic studies ase activity is stimulated by another cellular replication facto date, the process of DNA replication has been divided into tor, theproliferating cell nuclear antigen (PCNA) (Tsurimoto several distinct stages. The firststages, origin recognition and and Stillman, 1990). PCNA was first identified as a SV40 unwinding of the origin proximal DNA, require SV40 large replication factor required for coordinated leading and lagging tumor antigen (TAg), a cellular topoisomerase, and a cellular strand synthesis (Prelich et al., 1987a; Prelich and Stillman, protein called replication factor A (RF-A) (Wold et al., 1987; 1988). Under some circumstances, PCNA also stimulates the Tsurimoto et al., 1989;Borowiec et al., 1990). RF-A is a processivity of DNA polymerase 6 (pol 6), implicating this multisubunit replication factor comprising protein subunits polymerase in the replication of DNA. Indeed, several recent of relative molecular masses of70,000 (70 kDa), 34,000 (34 studies have directly demonstrated a role for DNA polymerase 6 in SV40 DNA replication (Lee et al., 1989; Weinberg and * This research was supported by Grant CA13106 from the NaKelly, 1989; Tsurimoto et al., 1990; Melendy and Stillman, tional Cancer Institute. The costs of publication of this article were defrayed in part by the payment of page charges. This article must 1991). therefore be hereby marked “adoertisernent” in accordance with 18 The three replication factors, RF-A, RF-C, and PCNA U.S.C. Section 1734 solely to indicate this fact. affect the activities of pol a and pol 6 in fundamentally The abbreviationsused are: TAg, SV40 large tumor antigen; RFA, replication factor A; RF-C, replication factor C; PCNA, prolifer- different ways. RF-A and RF-C both stimulate pol a activity ating cell nuclear antigen;pol a , D N A polymerase a-primase complex; under some circumstances, whereas RF-A, RF-C, and PCNA pol 6, DNA polymerase 6; Hepes, 4-(2-hydroxyethyl)-l-piperazineeth- cooperatively stimulate pol 6 activity (Kenny et al., 1989; anesulfonic acid. Tsurimoto and Stillman, 198913).

Replication factors A and C (RF-A and RF-C) and the proliferating cell nuclear antigen (PCNA) differentially augment the activities of DNA polymerases a and 6. The mechanism of stimulation by these replication factors was investigated using a limiting concentration of primed, single-stranded template DNA. RFA stimulated polymerase a activity ina concentrationdependent manner, but also suppressednonspecific initiation of DNA synthesis by both polymerases (Y and 6. The primer recognition complex, RF-C .PCNA. ATP, stimulated pol 6 activity in cooperation with RF-A, but also functioned to prevent abnormal initiation of DNA synthesis by polymerase a. Reconstitution of DNAreplication with purified factors and a plasmid containing the SV40 origin sequences directly demonstrated DNA polymerase a dependent synthesis of lagging strands and DNA polymerase G/PCNA/RF-Cdependent synthesis of leading strands. RF-A and the primer recognition complex both affected the relative levelsof leading and lagging strands. These results, in addition to results in an accompanying paper (Tsurimoto, T., and Stillman, B. (1991) J. Biol. Chem. 266, 1950-1960), suggest that an exchange of DNApolymerase complexes occurs during initiation of bidirectional DNA replication at the SV40 origin.

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The characterization of these proteins suggested functional similarities between the RF-C and PCNA factors and the bacteriophage T4 DNA polymerase accessory proteins encoded by genes 44/62 and gene 45, respectively (Tsurimoto and Stillman, 1990). Omission of either PCNA or RF-C from the replication reactions resulted in theaccumulation of short nascent strands thathybridized to thelagging strand template DNA (Prelich and Stillman, 1988; Tsurimoto and Stillman, 1989a). Furthermore, pol a and pol 6 synthesize short and long DNA strands, respectively, on singly primed singlestranded phage M13 DNA in the presence of various combinations of RF-A, PCNA, and RF-C(Tsurimoto and Stillman, 1989b). These results strongly suggested that during SV40 DNA replication, pol a and pol 6 synthesize the lagging and leading strands, respectively. The involvement of two separate DNA polymerases that appear to cooperatively replicate leading and lagging strands at the replication forkraises the question of how initiation of DNA replication occurs at the origin. For example, instead of a single initiation event for DNA synthesis, there must be at least two initiation events, one for lagging strand synthesis and one for leading strand synthesis. Recent studies have demonstrated sequential roles for pol a and pol 6 polymerase complexes in initiationof DNA replication from the SV40 origin (Tsurimoto et al., 1990). In an accompanying paper, we carried out mechanistic studies on the interaction of replication factors and DNA polymerases at aprimer-templatejunction(Tsurimoto and Stillman, 1991). Highly specific primer binding of a primer recognition protein complex (RF-C-PCNA) in cooperation with RF-A and dynamic assembly and disassembly of these components coupled with ATP hydrolysis have been demonstrated. In this paper, we have studied the primer-template junction recognition by DNA polymerase complexes via their interaction with the replication factors. A DNA polymerase switching model for initiation of leading strand DNA synthesis from an Okazaki fragmentsynthesized at the replication origin is demonstrated.Furthermore, we have reconstituted SV40 DNA replication in vitro with purified replication factors and two DNA polymerases based on this model. The mode of DNA synthesis is controlled by a balance of replication factor activities.

and theincorporated dAMP was measured by absorption of a sample of the reaction to DE81 filters. Product Analysis-The remaining portion of each reaction mixture from either DNA polymerase assays or SV40 DNAreplication assays wasmixed with an equal volume of astop mixture (0.2 mg/ml proteinase K, 2% sodium dodecyl sulfate, and 20 mM EDTA) and incubated at 37 "C for 30 min. DNA in a sample was extracted with phenol/chloroform (l:l), precipitated with ethanol, and subjected to electrophoresis in an alkaline agarose gel (1% or 2%) in 30 mM NaOH, 1 mM EDTA as described previously (Maniatis et al., 1982; Tsurimoto and Stillman, 1989b). After electrophoresis, the gel was fixed, dried, and autoradiographed. ReplicationFactors-Highly purified replication factors, RF-A, PCNA, RF-C, pol a-primase, pol 6, topoisomerases I and 11, and TAg were obtained by published procedures (Tsurimoto etal., 1989; Tsurimoto and Stillman, 1991). RESULTS

RF-A Affects DNA Synthesis by Pol a and 6"Nuclease footprinting experiments demonstrated that the binding of is pol a and pol 6.PCNA to aprimer-templatejunction affected by the amounts of RF-A present inthe reaction; most notably, RF-Ainhibits binding by pol 6 . PCNA and decreases binding by pol a (Tsurimoto and Stillman, 1991). Under some conditions, however, RF-A also functions as a stimulatory factor for both DNA polymerases on a primed templateDNA (Tsurimoto and Stillman, 1989b). These apparently contradictory results suggested that the stimulatory and inhibitory effects of RF-A may be dependent on its concentration. Indeed, an excess of primer-template DNA was used for previously reported DNA polymerase assays, but limitingamounts of the primer-template DNA were used in the footprinting assays, indicating that theratio of RF-A to DNA was different in the two experiments. To testthe effect of saturated amounts of RF-A in DNA polymerase reactions, the concentration of a primer-template DNA was decreased by 20-fold relative to the amount used in previous experiments (Tsurimot0 and Stillman, 1989b). As shown in Fig. lA, RF-A stimulated pol a activity, but at higher concentrations, incorporation was reduced. Analysis of the replication products by alkaline agarose gel electrophoresis revealed that pol a was poorly processive without RF-A (less than 20 nucleotides; Fig. 2, lane 1 ) but the length of the product was increased to near full length (an average of 300-400 nucleotides, Fig. 2, lanes 2 and 3 ) with MATERIALS AND METHODS RF-A present at 12.5-25 wg/ml. Interestingly, the length of DNA Polymerase Assays-A standard reaction mixture for DNA polymerase assays with poly(dA)/oligo(dT) template contained 30 the product was reduced to 100 to 200 nucleotides when higher amounts of RF-A were used (Fig. 2, lane 4 ) . Since the procmM HEPES, pH 8.0, 7 mM MgCl,, 0.5 mM dithiothreitol, 0.1 mg/ml bovine serum albumin, poly(dA) (average length 400), oligo(dT) (av- essivity of pol a in the presence of RF-A was measured to be erage length 12-15) (201; 4 PM nucleotide), and 0.05 mM [oI-~'P]TTP 100 to 200 nucleotides in previous experiments with higher (approximately 3,000 cpm/pmol). Note that the pH of these reactions concentrations of primed template DNA, the longer products is different from those used previously (Tan et al., 1986; Prelich et with an intermediate concentration of RF-A most likely were al., 198713) to demonstrate an effect of PCNA on the processivity of a result of re-initiation of DNA synthesis by pol a at the3'pol 6. The reaction mixture was incubated at 37 "C for 15 min, and one-fifth of the sample was spotted on DEAE-paper (DE81, What- end of a newly synthesized strand. Thus, inhibition of pol a man) to measure the incorporated radioactivity (Tsurimoto et al., activity by high amounts of RF-A was probably caused by 1990). The remainder of the sample was used for product analysis as suppression of reinitiation by pol a. described below. DNApolymerase assays with a singly primed, singleThe effect of RF-A on pol 6 activity was also determined. stranded DNA was carried out with the same reaction mixture except Pol 6 activity was reduced by the addition of RF-A, even in for the addition of 0.05 mM [w3'P]dATP (specificactivity 3,000 cpm/ the presence of either PCNA or RF-C (Fig. 1B). Theinhibipmol), 0.05 mM dGTP, 0.05 mM dCTP, and 0.05 mM TTP and 0.6 pg/ml single-stranded pUC118DNA primed with a 3-fold molar tion was substantially more than the reduction of pol a excess of a 17-base sequencing primer (primer 1211 from New Eng- activity. This was consistent with the results obtained by footprinting experiments which demonstrated that primerland Biolabs) instead of TTP and poly(dA)/oligo(dT). DNA Replication in Vitro with a Plasmid Containing the SV40 template recognition by the pol 6.PCNA complex was comReplication Origin"SV40 DNA replication in vitro wasassayed under pletely inhibited by RF-A, whereas the pol a complex was standard conditions as described previously (Tsurimoto et al., 1989). only partially reduced, Therefore, RF-A at high concentraComponents added were 80 pg/ml SV40 TAg, 4 Fg/ml calf thymus tions has a suppressing function for initiation of DNA syntopoisomerase I, 1.8 pg/ml calf thymus topoisomerase 11, 6.25 pg/ml thesis at the 3'-end of a primer by both pol 6 and pol 6, pSVOll DNA, and 87 units/ml calf thymus pol 6 (5.5 X lo3 units/ mg) and various amounts of RF-A, PCNA, RF-C, and pol a-primase although it functions as a processivity factor for pol a. DNA Synthesis by Pol 6 and the Primer Recognition Cornas indicated. The reaction mixture was incubated at 37 "C for 1 h,

FIG. 1. Effect of replication factors and ATP on DNA synthesis by pol a and pol 6. A, titration of the

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R. pol6

pol6 + PCNA+ RF-C

A. pol a

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amount of RF-A in DNA synthesis reactions containing pol n and poly(&)/ oligo(dT) as template-primer DNA. Reaction mixtures contained 3 pg/ml pol n and various amounts of RF-A. J? and C, titration of the amount of RF-A in DNA synthesis reactions containingpol 6 and poly(dA)/oligo(dT) as template-primer DNA.Reaction mixturescontained 26 units/ml pol 6, 6.7 pg/ml PCNA, 1 pg/ ml RF-C, and various amountsof RF-A as indicated. 1 mM ATP was added to reactionsin J? and +ATP in C. DNA synthesis was expressed as picomoles of TMP incorporated in a 2 5 4 reaction mixture following incuhation for 15 min.

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FIG. 3. DNA replication timecourse. A . effect of hlocking ATP hydrolysis during DNA svnthesis hv pol d on a singlv primed, singlestranded DNA in the presenceof RF-A, PCNA, andR F X . A reaction - 100 mixture containing 0.6 pg/ml single-stranded pC:(:llR DNA primed 100 with a sequencing primer (New England I{iolahs). 260 units/ml pol 6 , 70 pg/ml RF-A, 70 pg/ml PCNA. :I pg/ml IIF-C. and 0.1 m%i ATP was preincuhated at 0 "C for 5 min and then incuhated at :I7 'C. A t 30 - 30 time 2 min, ATP or A T P r S wasadded to themixtureto a final were concentration of 1 mM (as indicated),andtheincuhations continued at 37 "C. At each indicated time, an aliquot of the mixture sample was spottedontoDFAK-paperto was withdrawn,anda 567891011IE I 2 3 4 measure incorporation of Iahel. and another sample WRS analvzrd hv gel electrophoresis(see Fig. 4). DNA syntheRis was expressrd as FIG. 2. Products of DNA synthesis with pol a. Products ohtained from reactions containing pol n, as described in Fig. 1 and picomoles of dAMP incorporatedin a 25-pl reaction mixture. /j. cffect complex onpol n activity gel (2%)electrophoresis. of formation of the active primer recornition Table I, were subjected to an alkaline agarose using a singly primed. single-stranded template I)NA in the presence The single-stranded DNA marker wasfrom HpaII-digested, denatured pH13322 DNA run in parallel, and the length in nucleotides is of RF-A and ATP. A reaction mixture Containing 15 pg/ml pol 11. 70 pg/ml RF-A, 3 pg/ml RF-C. and 1 mM ATP wns preincuhated at 0 "C indicated. for 5 min and incuhated at 37 "C. At time 2 min. I'('NA was added to a final concentration of 70 pg/ml ( + I , or the same volume of huffer plex Coupled with ATP Hydrolysis-Previous experiments A (Tsurimoto P I nl., 19A9a) was added to themixture (-). Roth demonstrated thatpol 6 activity on a primed, single-stranded reactions continued to he incuhated at 37 "C. and DNA svnthesis and DNA template required RF-C, PCNA, RF-A, and hydrolysis product analysis at each time point were performrd as descrihed in 200

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A. of ATP (Tsurimoto and Stillman, 1989b). We therefore determined the effect of ATP on DNA synthesis by pol 6 in the presence of these factors. Fig. 1C shows a titration of increas- PCNA translocates along the DNA alone, or alternatively, of the polymerase complex. ing amounts of RF-A and its effect on pol 6 activity in the RF-C might remain an active part of primer- T o begin to address this question, we tested if the ATPase presence of RF-C and PCNA and limiting amounts of template DNA. In the absence of ATP, pol 6 activity was activity of RF-Cfunctionsduringtheelongationstage inhibited by increasing amountsof RF-A. If ATP was present, DNA synthesisfrom a primer-template junctionwith pol 6 by however, pol 6 activity was greatly stimulated. This suggests determining the effect of ATPyS on DNA synthesis. When RF-C and PCNA did not actively function as a primer rec- DNA synthesis by pol 6 in the presence of RF-A, RF-C, and primed template DNA was startedandthen ognition complex without ATP. Furthermore,when pol 6 was PCNAona correctly loaded onto the primed single-stranded DNA, RF-AATPyS was added after 2 min, DNA synthesis stopped com3A ). The addition pletely, immediat,ely after the addition (Fig. functioned as a stimulatory factor for DNA synthesis. of an excess of ATP, however, did not affect continued DNA The results of footprinting experiments (Tsurimoto and were analyzed by alkaline Stillman, 1991) demonstrate that pol 6.PCNA protected the synthesis.Whentheseproducts agarose gel electrophoresis, the extension of the nascent DNA same region of the primer-template junction as the active primkr recognition complex whichcontains RF-C. PCNA and strand also ceased immediately (Fig. 4 A ) . This result demATP. This raises the questionof the fate of RF-C once pol 6 onstrated that ATP hydrolysis is required to translocate the interacts with the primer. One scenerio is that only pol 6. pol 6 complex on a template. ATPyS did not have an effect

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FIG.4. Product analysis of DNA synthesis by pol a and pol 6. A, products of DNA synthesis by pol 6 on a singly primed, singlestranded DNA template. R, products of DNA synthesis by pol n on a singly primed, single-stranded DNA. In both cases, theproduct DNAs from reactions described in Fig. 3 were purified and subjected to alkaline agarose gel (1%) electrophoresis. The positions are indicated by the length of single-stranded DNA determined by electrophoresis of denatured adenovirus DNA that was digested with HindIII. ssl, is the position of a single-stranded linear template DNA (3.2 kilobases).

product synthesized under these conditions was 50-200 bases, which is the same as the products obtained by pol a and RFA alone (Fig. 2, lunes 5-8).Addition of RF-C slightly stimulated the incorporation, but significantly,full length product was produced (Fig. 2, lane 9 ) . This demonstrates that RF-C stimulates not only pol 6 but also pol a, as we have published 1989b).I t is not yetclear previously (Tsurimoto and Stillman, from these experiments whether RF-C increased the processivity of pol a directly or increased the efficiency of reinitiation of pol a on a previously synthesized nascentDNA strand. In contrast to these results, the effect of the active primer recognition complex (RF-C. PCNA-ATP) on pol a was completely different and opposite to the effect found on pol b. If the priming complex was in the inactive form lacking ATP, pol a exhibited the samemode of DNA synthesis aswith RFC alone (Table I).If, however, the complex was activated with ATP, pol a activity was strongly inhibited (Table I) and no products were detected on an alkaline agarose gel (Fig. 2,lune 12).

The same result was obtained by following a time course of DNA synthesis by pol a on a singly primed template DNA (Fig. 3). Pol a synthesized DNA constantly for a t least 14 min in the presence of RF-C and ATP, and relatively long DNA strandswere produced (Figs. 3R and 4R). If PCNA was TABLE I added to the reaction after DNA Synthesis for 2 min, this DNA synthesis with p o l n and RF-A on poly(dA)/oligo(dT) allowed RF-C and ATP toform the primer recognition comReaction mixtures contained 3 pg/ml pol a,50 pg/ml RF-A, 13.3 plex and DNA synthesis stopped immediately (Figs. 3R and pg/ml PCNA, 4 & n l RF-C,and 1 m M ATPas indicated. DNA 4R). This confirmed the result that the primer recognition synthesis was expressed as described in the legend for Fig. 1. complex blocked DNA synthesis by pol a, but also suggested Component added DNA synthesis that the elongation of DNA synthesis by pol a might be PCNA ATP RF-C regulated by PCNA or by the interaction of ATP with KF-C. pmol dTMP However, since the intrinsicprocessivity of pol a is low, this 10.8 experiment does not determine whether the active primer + 7.4 recognition complex blocks pol a translocation or whether it + 9.4 simply blocks reinitiation on preformed nascent DNA strands. + + 7.6 13.4 Reconstitution of SV40 Replication with Two DNA Polym+ erases-As shown above, initiation of DNA synthesis et the + + 12.0 13.4 + + 3'-end of a primer by pol a or 6 is controlled by two different + + + 2.2 mechanisms: the amount of RF-A and the formation of an active primerrecognition complex. We therefore investigated the effect of these two replication components onleading and on the low amount of DNA synthesis by pol 6 and PCNA on lagging strand synthesis during SV40 DNA replication. The the primed template DNA (data not shown),suggesting that results above and those recently obtained (Tsurimoto et al., the effect of ATPyS was RF-C-dependent. This suggests that RF-C translocates with the pol 6 during DNA synthesis, but 1990) suggest that initiation of DNA replication by pol n confirmation of this will require immunological reagents to results in the synthesis of the first Okazaki fragment at the detect RF-C, which are currently notavailable. In the analo- replication origin and then the primer recognition complex gous polymerase complex from bacteriophage T4, ATP hy- appears to be involved in a switching mechanism to remove drolysis by gene 44/62 protein complex is required for the pol a and load pol 6 onto the 3'-endof this Okazaki fragment assembly of the active DNA polymeraseholoenzyme a t a 3'- to initiate leading strand DNA synthesis. Thiscould explain was compromised by the absence end and is also required during the elongation stage (Huang why leading strand synthesis it does not explain why pol a of PCNA and RF-C; however, et al., 1981; Mace and Alberts, 1984). It has been proposed did not continue to self-prime and copy the leading strand that hydrolysis of ATP is involved in a timing mechanism that accounts for the recycling of DNA polymerase required template DNA, as hasbeen observed by Ishimi et al. (1988). T o address this point, and taking note of the effect of RFfor lagging strand DNA synthesis, since ATPyS did not block translocation of the DNA polymerase directly (Selick et al., A on pol a activity, we determinedwhether RF-A would 1987).Alternatively, it ispossible that ATPhydrolysis by the suppress nonspecific leading strand synthesis by pol a. Ingene 44/62 protein is required for translocation on both the creasing amounts of RF-A were added to reconstituted repliof essential leading andlagging strands, since ATPhydrolysis by RF-C is cation reactions containing various combinations replication factors. Inthefirstseries of experimentq,all required for leading strand synthesis by pol 6 . ATP-dependent Blocking of Pol a DNA Synthesis by the reactions contained a plasmid DNA harboring the SV40 repPrimer Recognition Complex-As showninTableI, DNA lication origin, TAg, topoisomerases I and 11, and pol a (Fig. synthesis by pol a in the presence of a high amount of RF-A 5A). In all cases, DNA replication absolutely required RF-A, was not significantly affected by PCNA or ATP, except for and the optimum amountof RF-A for incorporation was less slight inhibition by ATP. This inhibition with ATP seems tothan 12.5 pg/ml (Fig. 5A). In the absence of any additional was obtained replication components andwith 12.5 pg/ml RF-A concentrabe intrinsic to pol a, sincethesameresult without RF-A (data not shown). The length of the major tions, abimodal distribution of nascent DNA strands was

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previously reconstituted SV40 replication reactions containing six purified proteins and one crude fraction (Tsurimoto et al., 1989). Furthermore, this reconstituted replication system appeared, by several criteria, to reflect the mechanism of SV40 DNA replication observed with a crude lysate, except for the lack of production of mature ligated lagging strand replicated DNA (Tsurimoto et al., 1990). Most importantly, this purified replication system completely reproduced the requirement for RF-C and PCNA and directly demonstrated that the leading and lagging strands are synthesized by pol 6 and pol a, respectively. The Effect of Replication Factors on the Mode of SV40 DNA B. Replication-The reconstituted replication system was sensitive to the ratio of various components in addition to the amount of RF-A. As shown in Fig. 6B (lanes 1-4), short lagging strands were synthesized by pol a in the presence of high amounts of RF-A and in the absence of RF-C andPCNA (Figs. 5B and 6B, lanes 1-4). These fragments increased in length with increasing amounts of pol a, as reported previously ona singly primedtemplate DNA (Tsurimoto and 0 10 20 Stillman, 1989b). This suggests that high amounts of pol a pol a /primase (pg/ml) initiated DNA synthesis multiple times on the lagging strand L. template DNA. In the presence of the active primer recogni20 r tion complex (RF-C .PCNA. ATP),pol 6 was activated, but the amountof DNA replication was also dependent upon the amount of pol a (Fig. 5 B ) . Interestingly, withlow amounts of pol a, the short lagging strand DNA disappeared, but the longer leading strand DNA was synthesized (Fig. 6B, lunes 58 ) . At the lowest concentration of pol a,a significant amount of product equivalent to full length plasmid was produced 11 (Fig. 6B, lane 5). In the absence of enzymes that mature “ 0 0.5 1.0 Okazaki fragments into a continuous strand, this unitlength PCNA & RF-C DNA must beproduced by abnormal leading strand synthesis FIG. 5. Titration of replication factors in SV40 DNA repli- by pol 6 all the way around the template DNA, except for the cation reactions reconstituted with purified components. Replication reactions were performed ina 25-pl reaction mixtureat 37 “C initiation reaction by pol a at thereplication origin. Efficient for 60 min, and DNA synthesis was measured and is indicated as and coordinated leading and lagging strand DNA synthesis picomoles of dAMP incorporated. A, titration of RF-A. Components with purified proteins was seen in the presence of relatively used are indicated next to each line. Reactions contained 24 pg/ml high amounts of pol a (Fig. 6B, lane 8). With higher amounts pol a, 2 pg/ml RF-C, 6.7 pg/ml PCNA, 87 units/ml pol 6, and the of pol 6, more efficient DNA synthesis was obtained, but the amounts of RF-A indicated in the graph. B, titration of pola-primase product was changed from the bimodal distribution to almost complex. Components used are indicated next to each line. Reactions contained 50 pg/ml RF-A, 2.4 pg/ml RF-C, 8 pg/ml PCNA, and 87 all of the product migrating in the position of the unusual units/ml pol 6 and the amounts of pol a-primase complex indicated unit length leading strand (data not shown). Therefore, coin the graph. C, titration of the primer recognition complex (RF-C. ordinated leading and lagging DNA synthesis in uitro requires PCNA). Amount 1.0 represents 6 pg/ml RF-C and 20 pg/ml PCNA, balanced leading and lagging strand DNA polymerase activiand bothwerechanged to maintain this ratio. Other components ties. used are 18 pg/ml pol a, 50 pg/ml RF-A, and 87 units/ml pol 6 as The amountof the primer recognition complex also affects indicated in the graph. the type of DNA synthesis. When the complex was absent, detected by alkaline agarose gel electrophoresis (Fig. 6A, lune DNA replication with either pol a alone or pol a plus pol 6 1). These short and long DNAs correspond to thelagging and yielded similar products consisting of short, lagging DNA leading strand nascent DNAs, respectively, as has been re- strands (Fig. 6C, lanes 1 and 6 ) .However, addition of increasported by Ishimi et al. (1988). Increasing the amount of RF- ing amounts of the primer recognition complex suppressed A in the reaction decreased the incorporation slightly (Fig. DNA synthesis dependent upon pol a alone and shortened 5 A ) , but more importantly, this led to a dramatic elimination the product to 200-300 nucleotides in length (Figs. 5C and of the long nascent strands (Fig. 6A, lunes 2-4). Thus, RF-A 6C, lanes 7-10). In contrast, DNA synthesis in the presence modulates that activity of pol a by blocking nonspecific of both pol a and 6 was stimulated by the addition of optimal initiation on a leading strand template from the 3’-end of amounts of RF-C and PCNA, yielding a bimodal distribution Okazaki fragments. The addition of the primer recognition of DNA strands (Figs. 5C and 6 , lane 2). Higher amounts of complex (PCNA. RF-C) also blocked the long leading strands, the complex increased the population of the long products, even in the presence of low amounts of RF-A (Fig. 6A, lanes equal to or longer than unit length DNA strands (Fig. 6 , lane 5-9). This suggests that both RF-A and RF-C.PCNA limit 5 ) . This result again confirmed that the primer recognition pol a to the lagging strand template. The addition of pol 6, complex affected DNA polymerase switching for initiation of RF-C, and PCNA to the reactions restored the synthesis of leading strand DNA synthesis at the replication origin. Furthe long leading strands, and theirsynthesis became insensi- thermore, the concentration of the primer recognition comtive to high concentrations of RF-A (Fig. 6A, lanes 9-12). plex is an important factor for balancing the activity of the The amount of DNA replication obtained with these eight two polymerases, resulting incoordinated leading and lagging purified proteins was comparable to the levels obtained with strands synthesis. A.

loE

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Polo( POIS RNA

FIG. 6. Product analysis of the re- RF-c constituted SV40 DNA replication - RF-A reaction. The reaction products are derived from the experiments described in Fig. 5. Components used are indicated above each lane. Increasing amounts of RF-A in column A were 12.5, 25,37.5, SA and 50 pg/rnl. Increasing amountsof pol a-primase in column R were 6, 12, 18, and 24 pg/ml. Increasingamountsof RFC. PCNA incolumn C were 0,0.125,0.25, 0.5, and 1.0 (1.0 = 6 pg/ml RF-C and 20 pg/rnl PCNA). Marker positions are indicated by the length of single-stranded DNA obtained from denatured adenovirus DNA that had previouslybeen digested with HindIII. ssL is the position of a single-stranded linear template DNA (2.9 kilobases).

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As published previously, RF-A is a processivity factor for pol a (Tsurimoto et al., 1989b), but as described herein, it also blocks initiation of DNA synthesis by pol cy on the end of a nascent strand thatwas just synthesized. This latter function accounts for the suppression of nonspecific DNA synthesis from the 3'-end of Okazaki fragmentsby this polymerase. As shown in Fig. 6A, this suppressing functionof RF-A was also revealed during SV40 DNA replication in vitro, since high amounts of RF-A restricted DNA synthesis by pol cy to the lagging strand template. / .--"-I The results demonstrating an effect of RF-A on primed r""""""e!-?-!?! p"! b l :pola+ R A + p d b +high \$Nt DNA synthesis by pol a also presented a paradox. RF-A did not completely block DNA synthesis by pol a when primed FIG. 7. Three modes of DNA synthesis obtained during SV40 DNA replication with purified components. Type I, ab- by the unique oligonucleotide primer (the initial primer), but normal lagging strand DNA synthesis uncoupled from leading strand reinitiation at the3' end of the newly synthesized strandwas two potential synthesis. Type 11, lagging strand synthesis coupledwith unusual blocked by RF-A. This must mean that the leading strand synthesis by pol a alone, or coordinated leading and primers were not equally available to pol a. One interesting lagging strand synthesis by pol a plus pol b (complete). Type 111, explanation for this paradox is that a pol a-RF-A complex abnormal leading strand synthesis uncoupled from lagging strand might stably bind to the 3'-end of a newly synthesized strand synthesis. The conditions that lead to thesemodes of DNA replication and block reinitiation. This complex might then be released are summarized in thedashed boxes below. See text for details. by the combined actionof RF-C and PCNA, noting that RFC may indeed be a part of the lagging strand complex (TsurDISCUSSION imoto and Stillman, 1989b). Therefore, PCNA may play a A summary of the various modes of DNA replication ob- role in lagging strand DNA synthesis by cycling the lagging served in the presence of different amounts of replication strand polymerase complex from the 3'-end of a newly synfactors is schematically demonstrated in Fig. 7. Type I prod- thesized Okazaki fragment to a new location o n the lagging ucts result fromuncoupled lagging strand synthesis observed strand template for self-priming by primase. Such a mechanism is consistent with the results in Figs. 38 and 4R. This in the presence of high amounts of RF-A and when one or both of the components required for the leading strand polym-model has been suggested for phage T4 and E. coli lagging strand DNAreplication. This model also implies that the erase complexwas omitted. Type I1 productsresultfrom coordinated leading and lagging strand synthesis observed initial oligonucleotide primer cannot tightlybind pol n,as we have observed (Tsurimoto and Stillman,1991). Furthermore, with the optimized, completely reconstituted replication reaction or with low amounts of RF-A and pol a alone. Type during DNA replication, pol a would not normally need to p o l b complex. 111 products result from uncoupled leading strand synthesis encounter such preformed primers, unlike the RF-A also blocks initiation of DNA synthesis by pol b on a and were caused by either limited amountsof pol a or higher amounts of pol 6. In thiscase, only long, leading strandswere preformed primer. But a specific mechanism exist-q to load synthesized from the first Okazaki fragment and subsequent pol 6 onto the preformed primer thatrequires RF-C, PCNA, recognition complex is formed, pol lagging strand synthesiswas minimal. This study showed that and ATP. Once the primer DNA synthesis by the these three modes of DNA synthesis were interchangeable 6 binds to it, and RF-A then stimulates and depended upon the ratio of the replication components. leading strand polymerase complex. We suggest that this is Only with the optimized complete system, however, did the the mechanism for initiation of leading strand DNA replicamechanism of DNA replication reflect, by several criteria, the tion at the replication origin. mechanism of SV40 DNA replication obtained with the crude A Model for DNA Polymerace Switching during Initiation of Bidirectional DNA Replication-We have recently demonfraction in vitro and the mechanismobserved in vivo. strated that the initiation of DNA replication in vitro from RF-A Controls Primer Recognition by DNA Polymerases-

-

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SV40 DNA Replication in Vitro POI

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FIG. 8. DNA polymerase switching for initiation of leading strand synthesis atthe SV40 replication origin. In the presence of RF-A and DNA helicase (TAg), the SV40 origin is locally unwound to yield RF-A-coated single strands. Pol a-primase then synthesizes a primer RNA (the priming stage). The next stage shows synthesis of the first Okazaki fragment by pol aprimase in cooperation with RF-C. PCNA then recognizes the RF-C bound to the 3' end, thereby displacing pol aprimase and forming the active primer recognition complex. Pol a-primase then moves along the same DNA strand to the lagging strand template to synthesize the next Okazaki fragment. Pol 6 recognizes the active primer recognition complex, resulting ininitiation of leading strand synthesis. Only a helicase (TAg) moving with the lagging strand polymerase complex is shown, although it is likely that a 5' + 3' helicase functions in vivo. Waved lines indicate primer RNA. Thick and thin solid lines are template and nascent DNAs, respectively.

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the SV40 origin requires the sequential initiation by pol a , followed by pol 6. The pol a complex then moves from the origin to copy the lagging strand template, whereas the pol 6 complex copies the leading strand template. Taking these results and theresults from this and the accompanying paper (Tsurimoto and Stillman, 1991) into account, we propose a polymerase switching model for the initiation of leading strand synthesis at a replication origin which is shown in Fig. 8. Synthesis of a primer RNA and thefirst Okazaki fragment are carried out by pol a-primase following unwinding of the DNA by TAg, RF-A, and a topoisomerase (see Borowiec et al., 1990). During the Okazaki fragmentsynthesis, RF-C interacts with pol a, forming the lagging strand polymerase complex. There areseveral lines of evidence that support this idea. First, a complex containing RF-C and pol a was detected by a gel shift assay (Tsurimoto and Stillman, 1991). Secondly, RF-C stimulated pol a activity on a primed template DNA, even in the presence of high concentrations of RF-A, and stimulated lagging strand synthesis during SV40 DNA replication (data not shown). Third, as shown in Fig. 3B, addition of PCNA to a DNA synthesis reaction containing pol a,RFC , and ATP stopped polymerization immediately, suggesting that thelagging strand polymerase complex (pol a.RF-C) was preformed and that PCNA could modulate the extent of polymerization directly. RF-C, however, is dispensable for lagging strand synthesis, so it remains an open question as to the precise role played by RF-C during lagging strand DNA synthesis. After the synthesis of the first Okazaki fragment, PCNA

1 6'

leading strand synthesis

and ATP interact with the lagging strand polymerase complex and releases the pol a complex, forming the active primer recognition complex. Saturated amounts of RF-A also contribute to the arrest of DNA synthesis by blocking pol (Y and its release from the template. Once the primer recognition complex is formed, pol 6 efficiently recognizes the 3'-end of the first Okazaki fragment by direct protein-protein interactions resulting in an exchange of DNA polymerase from the lagging strand complex to the leading strand complex. The leading strand polymerase complex then translocates in a ATP-dependent manner along the template to continuously synthesize DNA. The transientlyreleasedpol a complex could bind to thelagging strand template and move away from the origin to synthesize Okazaki fragments, perhaps by cycling on the template in a PCNA-dependent manner (see Tsurimot0 and Stillman, 1991). Such coordinated replication of both strandsat a replication fork has been proposed by Alberts et al. (1982) and Kornberg (1982). This model is consistent with the data described in this report and in an accompanying paper (Tsurimoto and Stillman, 1991). It also draws upon the extensive biochemical analysis of the leading and lagging polymerase complexes from E. coli and phage T4, where the cycling of the lagging strand polymerase complex has been investigated (reviewed by McHenry, 1988). The novel feature of this model is the switching of DNA polymerase complexes that occurs at the replication origin to establish continuous leading strand synthesis. This model, however, could certainly apply to the initiation of bidirectional DNA replication that occurs at the

SV40 DNA Replication in Vitro Cloning. A Laboratory Manual, Cold Spring Harbor Laboratory, E. coli oriC and phage X replication origins, even though E. coli polymerase I11 functions tosynthesize both strands at the Cold Spring Harbor, NY McHenry, C. S. (1988) Annu. Reu. Biochem. 5 7 , 519-550 replication fork. Melendy, T., and Stillman, B. (1991) J. Bid. Chem. 266, 1942-1949 Acknowledgments-We wish tothank GlennBauer for critical reading of the manuscript, N. Kessler for excellent technical assistance, and B. Weinkauff for typing. REFERENCES Alberts, B. M., Barry, J., Bedinger, P., Formosa, T., Jongeneel, C. V., and Kreuzer, K. N. (1982) Cold Spring Harbor Syrnp. Quant. Biol. 47,655-668 Boroweic, J. A., and Hurwitz, J. (1988) EMBU J. 7,3149-3158 Borowiec, J. A., Dean, F. B., Bullock, P. A., and Hunvitz, J. (1990) Cell 60, 181-184 Brill, S. J., and Stillman, E. (1989) Nature 3 4 2 , 92-95 Challberg, M. D., and Kelly, T. J. (1989) Annu. Rev. Biochem. 5 8 , 671-717 Dean, F. B., Bullock, P., Murakami, Y., Wobbe, C. R., Weissback, L., and Hurwitz, J. (1987) Proc. Natl. Acad. Sci. U. S. A. 8 4 , 16-20 Dodson, M., Dean, F. B., Bullock, P., Echols, H., and Hurwitz, J. (1987) Science 238,964-967 Fairman, M. P., and Stillman, B. (1988) EMBO J. 7,1211-1218 Goetz, G. S., Dean, F. B., Hurwitz, J., and Matson, S. W. (1988) J. Biol. Chem. 263, 383-392 Huang, C.-C., Hearst, J. E., and Alberts, B. M. (1981) J. Biol. Chem. 256,4087-4094 Ishimi, Y., Claude, A., Bullock, P., and Hunvitz, J. (1988) J. Biol. Chem. 2 6 3 , 19723-19733 Kenny, M. K., Lee, S.-H., and Hunvitz, J. (1989) Proc. Natl. Acad. Sci. U. S. A. 86,9757-9761 Kenny, M. K., Schlegel, U., Furneaux, H., and Hunvitz, J. (1990) J. Bid. Chem. 2 6 5 , 7693-7700 Kornberg, A. (1982) Supplement to DNA Replication, W. H. Freeman and Co., San Francisco Lee, S.-H., Eki, T., and Hunvitz, J. (1989) Proc. Natl. Acad. Sci. U. S. A. 86, 7361-7365 Mace, D. C., and Alberts, B. M. (1984) J. Mol. Biol. 177,313-327 Maniatis, T., Fritsch, E. F., and Sambrook, J. (1982) Molecular

Prelich, G., and Stillman, B. (1988) Cell 5 3 , 117-126 Prelich, G., Kostura, M., Marshak, D. R., Mathews, M. B., and Stillman, B. (1987a) Nature 326,471-475 Prelich, G., Tan, C.K., Kostura, M., Mathews, M. B., So, A. G., Downey, K. M., and Stillman, B. (1987b) Nature 326,517-520 Roberts, J. M. (1989) Proc. Natl. Acad. Sci. U. S. A. 8 6 , 3939-3943 Selick, H. E., Barry, J., Cha, T.-A,, Munn, M., Nakanishi, M., Wong, M. L., and Alberts, 0. M. (1987) DNA Replication and Recombination. U C U Symposium on Molecular and Cellular Biology, pp. 183-214, Academic Press, Orlando, FL Stillman, B. (1989) Annu. Reu. Cell. Biol. 5, 197-245 Tan, C.-K., Castillo, C., So, A. G., and Downey, K. M. (1986) J. Biol. Chem. 261,12310-12316 Tsurimoto, T., and Stillman, B. (1989a) Mol. Cell. Biol. 9, 609-619 Tsurimoto, T., and Stillman,B. (1989b) EMBU J . 8,3883-3889 Tsurimoto, T., and Stillman, B. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 1023-1027 Tsurimoto, T., and Stillman, B. (1991) J. Biol. Chem. 266, 19501960 Tsurimoto, T., Fairman, M. P., and Stillman, B. (1989) Mol. Cell. Biol. 9, 3839-3849 Tsurimoto, T., Melendy, T., and Stillman, B. (1990) Nature 346, 534-539 Weinberg, D. H., and Kelly, T. J. (1989) Proc. Natl. Acad. Sci. U. S. A. 86,9742-9746 Wiekowski, M., Schwarz, M. W., and Stahl, H. (1988) J . Biol. Chem. 263,436-442 Wobbe, C. R., Weissbach, L., Borowiec, J. A,, Dean, F. B., Murakami, Y., Bullock, P., and Hunvitz, J. (1987) Proc. Natl. Acad. Sci. U. S. A. 8 4 , 1834-1838 Wold, M. S., and Kelly, T. (1988) Proc. Natl. Acad. Sci. U. S. A. 85, 2523-2527 Wold, M. S., Li, J . J., and Kelly, T. J. (1987) Proc. Natl. Acad. Sci. U. S. A. 84, 3643-3647 Wold, M. S., Weinberg, D. H., Virshup, D. M., Li, J. J., and Kelly, T. J. (1989) J. Bid. Chem. 2 6 4 , 2801-2809