Proc. Nati. Acad. Sci. USAVol. 83, pp. 2869-2873, May 1986Biochemistry

Role of DNA polymerase a and DNA primase in simian virus 40DNA replication in vitro

(large tumor antigen/eukaryotic DNA replication)

YASUFUMI MURAKAMI, C. RICHARD WOBE, LAWRENCE WEISSBACH, FRANK B. DEAN,AND JERARD HURWITZGraduate Program in Molecular Biology and Virology, Sloan-Kettering Institute for Cancer Research, New York, NY 10021

Contributed by Jerard Hurwitz, December 30, 1985

ABSTRACT The role of DNA polymerase a (pol a) andDNA primase has been investigated in the simian virus 40(SV40) DNA replication system in vitro. Removal of pol a andprimase activities from crude extracts of HeLa cells or monkeycells by use of an anti-pol a immunoaffinity column resulted inthe loss of replication activity. The addition of purified pola-primase complex isolated from HeLa cells or monkey cellsrestored the replication activity of depleted extracts. In con-trast, the pol a-primase complex isolated from either mousecells or calf thumus did not. Extracts prepared from mousecells (a source that does not support replication ofSV40) did notreplicate SV40 DNA. However, the addition of purified pola-primase complex isolated from HeLa cells activated mousecell extracts. pol a and primase from HeLa cells were exten-sively purified and separated by a one-step immunoaffmityadsorption and elution procedure. Both activities were re-quired to restore DNA synthesis; the addition of pol a orprimase alone supported replication poorly. Crude extracts ofHeLa cells that were active in SV40 replication catalyzed thesynthesis of full-length linear double-stranded (RFIII) DNA inreaction mixtures containing poly(dT)-tailed pBR322 RFIII.Maximal activity was dependent on the addition of oligo(dA),ATP, and creatine phosphate and was totally inhibited byaphidicolin. Since pol a alone could not replicate this substrateand since there was no degradation of input DNA, we proposethat other enzymatic activities associate with pol a, displace thenon-template strand, and allow the enzyme to replicate throughduplex regions.

The replication of simian virus 40 (SV40) DNA is an impor-tant model system for studying eukaryotic DNA replicationbecause it requires only one virus-encoded protein, the SV40large tumor antigen (T antigen); all the other componentsinvolved in this process are supplied by host cells. Ourinterest is in the isolation of factors that are involved inmammalian DNA replication. For this purpose, we havedeveloped an in vitro replication system that is analogous tothose described by others (1-3), using a salt extract ofexponentially growing HeLa cells and plasmid DNA con-taining the SV40 origin sequence (4, 5). Replication withthese extracts was shown to be totally inhibited byaphidicolin, a specific inhibitor ofDNA polymerase a (pol a).In this communication we describe the role of pol a and DNAprimase in the replication system. pol a plays a key role inmammalian DNA replication (6-8). We have found that pola and primase activity are essential for the in vitro replicationofDNA containing the SV40 origin. We have also found thatthe cellular source of these activities plays an important rolein determining whether SV40 DNA replication can becatalyzed by extracts in vitro. Those cells that support

replication in vivo yield extracts that replicate SV40 DNA invitro; cells that do not support replication yield extracts thatare inactive. The pol a-primase complex isolated frompermissive cells supported replication in extracts depleted ofthese activities, whereas the complex isolated from nonper-missive cells was inactive.

In addition, we have developed a replication assay withwhich we can independently study the elongation step ofDNA replication using linear duplex DNA with long single-stranded tails at its 3' termini.

MATERIALS AND METHODS

In Vitro Replication System for SV40 ori+ DNA. Thereaction conditions and methods used for the preparation ofextracts of HeLa cells (4) and SV40 T antigen (9) have beendescribed. The extracts from FM3A mouse cells and monkeyCOS cells were prepared the same way as extracts from HeLacells. Reaction mixtures (50 Al) contained 30 mM Tris HClbuffer (pH 8.5); 7 mM MgCl2; 0.5 mM dithiothreitol; 4 mMATP; 200 AM each CTP, UTP, and GTP; 100,uM each dATP,dCTP, and dGTP; 25 tkM [methyl 3H]dTTP (300 cpm/pmol);40 mM creatine phosphate; 1 ,pg of creatine kinase, 0.3 Ag ofsuperhelical circular duplex (RFI) plasmid (pSV01AEP3,ori+); 200-400 Ag ofHeLa extract; and 0.5-1,4g ofT antigen.The ori+ DNA contained the 311-base-pair (bp) origin ofreplication (the EcoRII fragment G of SV40 DNA) insertedinto a 2481-bp pBR322 derivative as described (4). Reactionmixtures were incubated at 370C as indicated and the radio-activity incorporated into acid-insoluble material was mea-sured. When products obtained in the replication reactionwere analyzed, [a-32P]dCTP (3000 cpm/pmol) was used andanalysis was carried out by agarose gel electrophoresisfollowed by autoradiography.

Depletion of pol a-Primase Complex. An anti-pol a anti-body column was prepared using SJK-287-37 hybridoma[American Type Culture Collection (10)] supernatant andanti-mouse IgG-Sepharose as described (11, 12). The culturesupernatant (300 ml) was used to prepare a 5-ml antibodycolumn. A crude extract (100 mg of protein) from HeLa cells(4) was adjusted to 0.35 M NaCl and applied to a 2-ml column.The flow-through fraction was dialyzed against buffer A [20mM Tris HCl, pH 8.5/1 mM EDTA/1 mM dithiothreitol/10%(vol/vol) glycerol/50 mM NaCl] and was used as the pola-primase-depleted extract.

Purification of pol a-Primase Complex. A crude extractfrom HeLa cells (4) (prepared from 1010 cells), adjusted to 250mM NaCl in buffer B (20 mM Tris-HCl, pH 8.5/1 mMEDTA/1 mM dithiothreitol/20% glycerol), was applied to a

Abbreviations: SV40, simian virus 40; T antigen, SV40-encodedlarge tumor antigen; RFI, superhelical circular duplex DNA; RFII,circular duplex DNA containing at least one single-strand break;RFIII, linear product formed from RFI DNA; pol a, DNA polymer-ase a; u, unit(s).

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DEAE-cellulose column (100 ml) equilibrated with buffer Bplus 250 mM NaCl. The flow-through fraction was dialyzedagainst buffer B containing 50 mM NaCl and applied to asecond DEAE-cellulose column (50 ml) equilibrated withbuffer B containing 50mM NaCl. The pol a-primase complexwas eluted by the addition of buffer B containing 250 mMNaCl. The eluate (32 ml) was directly applied to aphosphocellulose column (50 ml) equilibrated with buffer Bplus 150 mM NaCl. The column was subjected to gradientelution (0.15-1 M NaCl linear gradient, 300 ml). pol a andprimase were co-eluted between 300 mM and 500 mM NaCl.The active fractions were pooled and dialyzed against bufferB plus 50 mM NaCl and loaded onto a single-strandedDNA-cellulose column (20 ml) prewashed with buffer Bcontaining 50 mM NaCl. The column was developed with a120-ml linear gradient of 50-500 mM NaCl in buffer B; theenzyme activities were recovered between 75 mM and 150mM NaCl. Active fractions were directly applied to a 1-mlphosphocellulose column and eluted with 4 ml of buffer Bcontaining 1 M NaCl. The eluate was dialyzed against bufferB containing 50 mM NaCl and used as the purified complex.Mouse pol a-primase complex was purified from 6 x 109 cells[mammary carcinoma FM3A c128 (13)] and monkey pola-primase complex was purified from 109 COS-1 cells, usingthe procedure described above. The specific activities ofthese enzymes in the assays described below were as follows:preparations from HeLa cells, 0.20 unit (u) of pol a and 0.50u of primase per ug of protein; from FM3A cells, 0.60 u of pola and 0.41 u of primase per Atg; from COS-1 cells, 0.25 u ofpol a and 0.55 u of primase per ,ug. Calf thymus enzyme waspurchased from Pharmacia and contained 0.14 u of pol a and0.05 u of primase per Ag.

Separation of pol a and Primase. The separation of HeLacell primase activity and pol a was carried out as follows.Crude extract (75 mg of protein) of HeLa cells (4), adjustedto 0.35 M NaCl in buffer C (20 mM Tris HCl, pH 8.5/1 mMEDTA/1 mM dithiothreitol/10o glycerol), was applied to a2-ml antibody column, containing anti-pol a monoclonalantibody (from hybridoma SJK-237) and equilibrated withbuffer C plus 0.35 M NaCl. After washing with 6 ml of thestarting buffer, both activities were eluted separately by thestepwise addition of 37% and 50%6 (vol/vol) ethylene glycolin buffer C plus 0.5 M NaCl (6 ml each). The separatedfractions were dialyzed against buffer C containing 50 mMNaCl. This procedure yielded primase (6 u/,ug of protein) andpol a (20 u/pug of protein) preparations that were virtually freeof cross-contamination (<2%).The mouse pol a-primase complex was resolved into its

individual activities (14) as follows. The purified complex (4ml), prepared as described above, was dialyzed against bufferC containing 50% ethylene glycol and 50 mM NaCl and thenwas applied to a 2-ml DEAE-cellulose column equilibratedwith buffer C containing 50%o ethylene glycol and 0.25 MNaCl. The primase activity was recovered in the flow-through fraction, and pol a activity was eluted by thestepwise addition of 6 ml of buffer C containing 50%6 ethyleneglycol and 0.25 M NaCl. The specific activity of each enzymedid not change in this step.Assay systems for pol a and primase have been described

(15). The reaction mixture (50 gl) for the pol a assaycontained 50 mM Tris HCl (pH 7.9); 7.5 mM MgCl2; 4 mMdithiothreitol; 20 pug of bovine serum albumin (BSA); 40 ILMeach dATP, dCTP, and dGTP; 8 uM [3H]dTTP (100cpm/pmol); and 10 ,g of activated calf thymus DNA (16).The reaction mixture (50 ul) for the primase assay contained50 mM Tris HCl (pH 7.9); 4 mM dithiothreitol; 9 mM MgCl2;10 ,ug of BSA; 1 nmol of poly(dT) (P-L Biochemicals); 8 gM[3H]dATP; 5 mM ATP; and 0.5 u of DNA polymerase I(Boehringer Mannheim). One unit of pol a and one unit of

primase incorporated 1 nmol ofdTMP and 1 nmol of dAMP,respectively, after incubation at 300C for 30 min.

Preparation of Poly(dT)-Tailed pBR322 RFIII. pBR322 RFIDNA was linearized with Pvu I (New England Biolabs) underthe supplier's recommended conditions and 0.2 volume of "5x TdT tailing buffer" (Bethesda Research Laboratories), 50.aM [methyl-3H]dTTP (Amersham), and 22 u of terminaldeoxynucleotidyltransferase (Pharmacia) were added. Incu-bation was continued at 370C for 4 hr. The DNA was thenextracted with phenol/chloroform (1:1), ethanol-precipita-ted, dissolved in 10 mM Tris Cl, pH 8.0/1 mM EDTA, andstored at 40C. An average of 250 pmol of dTMP wasincorporated per pmol of 3' terminus.

RESULTSThe Role of pol a and Primase in SV40 orl' Replication. In

order to study the role of pol a and primase, pol a-primase-depleted extracts were prepared by passing the crude extractfrom HeLa cells through an anti-pol a immunoaffinity col-umn. This procedure removed about 70% ofpol a activity and90% of primase activity present in the extract. Furtherrecycling of the depleted extract through i-nmunoaffinitycolumns did not remove the residual pol a activity. When thedepleted extract was examined in the SV40 replicationsystem, it was inactive (Fig. LA). This suggested that the pola-primase complex is essential for replication. This wasverified by the reconstitution of the replication activity withpurified pol a-primase complex isolated from HeLa cells(Fig. 1A). The extent of replication depended upon theamount of pol a-primase activity added; all incorporationwith the reconstituted system required T antigen and DNAcontaining the SV40 origin. The pol a-primase complexpurified from monkey COS cells was as active as that fromHeLa cells. In contrast, the complex purified from mouseFM3A cells or calf thymus did not restore the replicationactivity (Fig. LA). The combination of the pol a-primase

i 30 -E

.a

'oca 200

a-I-_ Q

20

0.4 0DNA polymerase a added(unit)

FIG. 1. Reconstitution of DNA replication by pol c-primase-depleted HeLa cell extract by the addition of pol a-primase complexisolated from various sources. (A) Reconstitution using HeLa celldepleted extract. Various amounts of pol a-primase complex fromHeLa cells (s), FM3A mouse cells (A), COS cells (v), and calfthymus(m) were added to the pol a-primase-depleted extract of HeLa cells.Incubations were carried out in the presence of T antigen (solidsymbols) or in the absence ofT antigen (open symbols) for 90 min at370C, and the amount of acid-insoluble radioactivity was determined.In each assay, 250 jug of protein was used. Under these conditions,the original HeLa cell extract catalyzed the incorporation of 76.8pmol ofdTMP after 90 min at 370C. (B) Reconstitution using COS celldepleted extract. Conditions were as in A, except that the pola-primase-depleted extract (250 jg ofprotein per assay) was isolatedfrom COS cells. Symbols used are the same as ih A. Under theseconditions, the COS cell extract prior to depletion catalyzed theincorporation of 34 pmol of dTMP.

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complex from thymus or mouse cells with the complex fromactive sources did not inhibit the latter. The pol a and DNAprimase activities in extracts from monkey cells were alsoremoved by immunoaffmity columns. These depleted mon-key extracts supported SV40 replication when supplementedwith the pol a-primase complex isolated from either HeLacells or monkey cells (Fig. 1B).

Activation of Mouse Cell Extract by HeLa pol ai-PrimaseComplex. The experiments described above were carried outwith the pol a-primase-depleted extract from cell extractsthat supported SV40 replication. Monkey cells and humancells are known to be permissive for the DNA replication ofSV40 in vitro (17) as well as in vivo (18). In contrast, mousecells are nonpermissive for DNA replication of SV40 but arepermissive for the DNA replication of polyoma virus. Todetermine what factor(s) is responsible for the permissive-ness in the SV40 DNA replication in vitro, we preparedextracts from mouse FM3A cells and tested various fractionsobtained from HeLa cell extracts. As shown in Fig. 2,purified pol a-primase complex isolated from HeLa cellsactivated mouse cell extracts which were inactive by them-selves. The addition ofmouse pol a-primase complex had noeffect. The activation was dependent upon the presence ofTantigen and DNA containing the SV40 origin (Fig. 3, lanes 10and 11). In addition, replication products formed were thesame as those synthesized by extracts of HeLa cells (Fig. 3,lanes 4 and 12). These results suggest that the pol a-primasecomplex plays an important role in the determination ofpermissiveness of SV40 DNA replication in vitro. This isconsistent with earlier results showing permissiveness is dueto trans-acting factors required for viral DNA replication ofSV40 (19).

Reconstitution of Replication Activity with Separated pol caand Primase Activities. As described in the previous section,the replication reaction with depleted extract was restored bythe addition of the HeLa cell pol a-primase complex. Weexamined whether both pol a and primase activities wereessential for replication. For this purpose, HeLa pol aactivity was separated from primase activity by use of ananti-pol a antibody column. After the application ofthe HeLacrude extract to the column, primase and pol a were elutedindividually (Fig. 4). Both enzymes were highly purified bythis one-step procedure. These isolated activities were addedto depleted extracts and assayed for replication activity(Table 1). The combination of HeLa cell pol a and Hela cellprimase supported replication, whereas the addition of eitherenzyme alone did not. These results suggest that both pol a

20r

-aE0.-o0

00.0.0u

a-

10

0C0 1.0 2.DNA polymerase a added (units)

RFII(orifl -

RFI tori-)-

1 2 3 4 5 6 7 8 9 10 1112j | ~~~~~ | -~~-Origin

11 i7-RFTII or:+)

-a

* ~ ~~~~-RFIkori+)l

FIG. 3. Analysis of DNA products formed by various cellextracts and HeLa cell pot a-primase. Reaction mixtures containedHeLa cell extract (lanes 1-4) or mouse cell extract (lanes 5-12) with(lanes 2, 4, 6, 8, 10, and 12) or without (lanes 1, 3, 5, 7, 9, and 11) SV40T antigen, using DNA with (lanes 3, 4, 7, 8, 11, and 12) or withoutthe SV40 origin sequence (lanes 1, 2, 5, 6, 9, and 10). Halfthe reactionmixtures containing mouse cell extract (lanes 9-12) were supple-mented with HeLa pol a-primase (1 u of pol a and 2.5 u of primase).RFI (replicative form I), superhelical circular duplex DNA; RFII,circular duplex DNA containing at least one single-strand break.

and primase activities are essential for replication. We alsocarried out the reconstitution experiment using mouse pol aand mouse primase in conjunction with the HeLa enzymes.None of the mouse enzymes worked when complementedwith the HeLa enzyme (Table 1). This suggests that both thepol a and the primase from a permissive cell are essential forreplication. Neither mouse pol a nor mouse primase prepa-ration inhibited the replication carried out with the HeLa cellenzymes (unpublished results), thus demonstrating the ab-sence of inhibitors in the mouse fractions.

Replication of Poly(dT)-Tailed pBR322 RFM. The initiationreaction with SV40 ori+ DNA is dependent on the T antigen.Primase and pot a play an important role in this event.However, once DNA chains have been started, pot a mustparticipate in an elongation reaction. Although pot a canextend 3'-hydroxyl ends hydrogen-bonded to a single-strand-ed DNA template, it cannot extend primer ends that arejuxtaposed to a duplex structure. These findings suggest thatauxilliary factors must act conjointly with pot a.The fractions that support SV40 replication were examined

for their ability to support elongation of primed templatesthrough duplex DNA. For this purpose, we added poly(dT)chains at each 3' terminus of pBR322 RFIII DNA. Underconditions similar to those used for the in vitro replicationreaction (see above), HeLa extracts catalyzed the incorpo-ration of dCMP into pBR322 RFIII that contained single-

_ 2000E0.

-o0)

0I-

°1000U

a.I-'o,.0

FIG. 2. Activation of mouse cell extracts with purified pola-primase. Various amounts of purified pol a-primase complex fromHeLa (0, o) or mouse FM3A (*, o) cells were added to mouse FM3Aextract (250 ,4g of extract protein). Reactions were carried out in thepresence of T antigen (solid symbols) or in the absence of T antigen(open symbols). After a 2-hr incubation, acid-insoluble radioactivity*was determined.

20 30 40Fraction Number

10 IE0.

-O0

5 E.°00

a.4X

60

FIG. 4. Separation ofHeLa pol a-primase complex into pol a andprimase fractions. HeLa cell crude extract (75 mg of protein) wasapplied to an anti-pol a column. Primase (e) was eluted with 37%ethylene glycol in buffer C and pol a (o) was eluted with 50% ethyleneglycol in buffer C. Fractions 17-21 and fractions 46-53 were pooled,dialyzed, and used as such.

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Table 1. Reconstitution of the replication activity with pol a andprimase from HeLa cells or from mouse FM3A cells

[3H]dTMPincorporated,

Enzyme(s) added pmol

HeLa pol a (0.4 u) 1.25HeLa primase (0.5 u) 0.36HeLa pol a (0.4 u) + HeLa primase (0.5 u) 16.7FM3A pol a (0.4 u) 0.80FM3A primase (0.5 u) 0.05HeLa pol a (0.4 u) + FM3A primase (0.5 u) 0.60FM3A pol a (0.4 u) + HeLa primase (0.5 u) 0.60FM3A po1 a (0.4 u) + FM3A primase (0.5 u) 0.05

Reaction mixtures (50 tll) were as described in Materials andMethods, with 0.3 ,ug of pSVO1AEP (ori'DNA), 250 ug of pola-primase-depleted HeLa, and 0.5 /ig of T antigen. The amount ofpurified enzyme(s) added to the depleted extract is indicated inparentheses. Acid-insoluble radioactivity was determined after 2 hrof incubation at 370C. The depleted extract incorporated 1.21 pmolunder the conditions described above. This number has beensubtracted from those presented above.

stranded poly(dT) tails at its 3' termini. This reaction wasdependent upon the presence of the poly(dT) tails and wasmarkedly stimulated by the addition of oligo(dA) primers(Table 2). The reaction was inhibited by 0.1 mM aphidicolin[a concentration sufficient to selectively inhibit pol a (20,21)], but purified pol a-primase alone could not catalyze thisreaction. ATP and an ATP-regenerating system were alsorequired (Table 2). The poly(dT) tails were not degraded(<5% of [3H]dTMP was recovered in acid-soluble materialafter a 30-min incubation), nor was the 32P-labeled DNAproduct after phenol extraction and reincubation with theHeLa extract (unpublished observations).

Polyacrylamide gel electrophoresis under denaturing con-ditions showed that the products were apparently full-length(Fig. 5, lane 1). To determine whether this was due to repairsynthesis, the DNA was cut with Dpn I, which will cleaveonly if both adenosine residues in its recognition sequence(GATC) are methylated. Since the pBR322 DNA was main-tained in a dam' strain ofEscherichia coli (HB101), productsarising from short-patch repair-type synthesis would be

Table 2. Requirements for replication of poly(dT)-tailed pBR322RFIII

1 23Origin-

RFM- X!

-1007

662

-337

-204

-196

-90

-55

FIG. 5. Analysis of product formed in replication assay usingpoly(dT)-tailed pBR322 DNA. Replication assays were carried out asdescribed in the legend to Table 2; terminated by the addition of0.5%NaDodSO4, 10 mM EDTA, and 10 jg of yeast tRNA; and digestedwith proteinase K (200 ,jg/ml) for 30 min at 370C. Products were thenisolated by phenol extraction and ethanol precipitation, collected in80% formamide, heated to 90°C for 3 min, and, after the treatmentsgiven below, subjected to electrophoresis in a 3.5% polyacrylamidegel [30:1 (wt/wt) acrylamide/N, N'-methylenebis(acrylamide)] con-taining 8 M urea. Lanes: 1, no treatment; 2, digestion with 1 unit ofSau3Al; 3, digestion with 1 unit ofDpn I. Restriction digestions wereperformed under the suppliers' recommended conditions. Prior toelectrophoresis, the restriction enzyme-digested samples were phe-nol-extracted and ethanol-precipitated a second time. Numbers atright indicate the length of DNA markers in base pairs.

degraded; products of full-length replication should remainintact. As shown in Fig. 5, lane 3, the products were largelyresistant toDpn I but sensitive to Sau3Al (lane 2), which cutsat the same sequence but is insensitive to the methylationstate of the DNA. The products were resistant to Mbo I(unpublished observation), an enzyme that cleaves only fullyunmethylatedGATC sites, indicating that progeny moleculeswere not utilized for multiple rounds of replication.

Conditions

CompleteOmit extractOmit DNAOmit oligo(dA)Omit DNA, add pBR322 RFIII

without poly(dT) tailsOmit extract, add pol

a-primase complexOmit ATP, creatine

phosphate, and creatinekinase

Add aphidicolin (0.1 mM)

dCMP incorporated,fmol/30 mi

363<13

86

36

20

138

Complete reaction mixtures (25 tl) contained 30 mM Tris-HCL(pH 8.5); 7 mM MgCl2; 0.5 mM dithiothreitol; 4 mM ATP; 200 ,uMCTP, GTP, and UTP; 100 AiM dATP, dCTP, and dTTP; 0.5 jiM

[a-32P]dCTP (800 cpm/fmol, Amersham); 40 mM creatine phos-phate; 1 jig of creatine kinase; 50 ng of poly(dT)-tailed pBR322RFIII; 50 pmol of (dA)1218 (P-L Biochemicals); and 100 jg (protein)of HeLa cell extract. After incubation for 30 min at 37TC, acid-insoluble radioactivity was measured. Where indicated, HeLa pola-primase complex (1 u of pol a and 2.5 u of primase) was added.

DISCUSSIONWe have used an anti-pol a monoclonal antibody to analyzethe role of pol a and primase in the replication of DNAcontaining the SV40 origin. Since primase forms a tightcomplex with pol a in higher eukaryotes (15, 22), thisprocedure also removed primase. The depleted extracts wereinactive in the replication reaction. The addition of purifiedpol a-primase complex restored this activity. In contrast, pola devoid of primase activity did not support replication;primase, containing no pol a, also did not restore replicationactivity. The combination of pol a and primase activitiesworked well, indicating that both enzymes are essential forreplication.The depleted system enabled us to test different pol

a-primase-complex preparations for their ability to supportreplication. Pol a-primase complex purified from exponen-tially growing mouse cells and from calf thymus, neither ofwhich is a natural host for the propagation of SV40 virus,were inactive, suggesting that the enzymes from cells that cansupport replication of SV40 DNA are required. In accordwith this idea, the pol .-primase complex purified frommonkey cells, a natural host for SV40, could reactivate the

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depleted extract. Human cells are not natural hosts for SV40virus, but they are permissive for replication of SV40 DNA(17, 18). Therefore, it is possible that the permissiveness ofa given cell centers on the ability ofT antigen to interact withthe cellular pol a-primase complex, as well as on the abilityof pol a-primase to interact with other cellular components.To test this possibility, we tried to activate mouse cellextracts (inactive by themselves) with various fractionsobtained from HeLa cell extracts. pol a-primase-depletedextract failed to activate mouse cell extract (unpublishedresult). However, the addition of purified HeLa cell pola-primase fractions to mouse extracts resulted in the repli-cation of SV40 ori+ DNA. This suggests that the pola-primase complex plays a key role in determining whethera particular cell supports SV40 replication. It also suggeststhat most of the replication factors in the mouse-cell extractcan work in the in vitro replication of SV40 DNA. The findingthat pol a and primase must both be derived from a permis-sive cell suggests that the specificity resides in the complex.

In addition to the replication assay described above, wehave also developed an elongation assay utilizing linearduplex DNA with primed single-stranded tails, a substrateresembling the leading strand of a replication fork. Nucleo-tide incorporation with this substrate was not as efficient aswith the SV40 replication system. This elongation reactionwas clearly dependent upon the presence of single-strandedDNA and an oligonucleotide primer, and the sensitivity toaphidicolin suggests that pol a is involved. Since pol a alone(or with primase) is incapable of replicating through duplexregions ofDNA (Table 2 and ref. 23), the extract must con-tain factors that can remove this impediment, either nucleo-lytically or by strand-displacement. Since the substrateDNA, as well as the products, was not degraded under ourreplication conditions (see Results), we suggest that the latteris the case. Similar substrates have been used to study theability of E. coli helicase II (24) and the adenovirus DNA-binding protein (unpublished results) to stimulate replicationby their cognate DNA polymerases, presumably by strand-displacement.

In this communication, we have identified pol a andprimase as factors that are essential for SV40 DNA replica-tion in vitro. In addition to these proteins, we have resolvedmultiple fractions, all of which are required for replication(11). It is clear that the SV40 system is amenable to purifi-cation and will provide important insights into chromosomalreplication.

Note Added in Proof. We have recently developed an in vitro systemthat supports the replication of polyoma virus DNA in the presenceof polyoma large T antigen. In this system, extracts of HeLa cellswere inactive, whereas extracts of mouse cells fully supported DNA

replication (Y.M., T. Eki, C. Prives, M. Yamada, and J.H., unpub-lished observations).

We are indebted to Ms. Claudette Turck and Ms. Nilda Belgado fortheir technical assistance. This work was supported by GrantGM34559 from the National Institutes of Health. C.R.W. is aPostdoctoral Fellow of the Damon Runyon Foundation and L.W. isa Postdoctoral Fellow of the National Institutes of Health.

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