Proc. Natl. Acad. Sci. USAVol. 90, pp. 5086-5090, June 1993Biochemistry

The El protein of bovine papilloma virus 1 is an ATP-dependentDNA helicaseLiu YANG, IAN MOHR*, ERIK FOUTS, DANIEL A. LIM, MICHAEL NOHAILE, AND MICHAEL BOTCHANtDepartment of Molecular and Cell Biology, 401 Barker Hall, University of California, Berkeley, CA 94720

Communicated by Nicholas R. Cozzarelli, March 5, 1993

ABSTRACT For efficient DNA replication of papilloma-viruses, only two viral-encoded proteins, El and E2, arerequired. Other proteins and factors are provided by the hostcell. E2 is an enhancer ofboth transcription and replication andis known to help El bind cooperatively to the origin of DNAreplication. El is sufficient for replication in extracts preparedfrom permissive ceils, but the activity is enhanced by E2. Herewe show that purified El can act as an ATP-dependent DNAhelicase. To measure this activity, we have used strand dis-placement, unwinding of topologically constrained DNA, de-naturation of duplex fragments, and electron microscopy. Theability ofEl to unwind circularDNA is found to be independentof origin-specific viral DNA sequences under a variety ofexperimental conditions. In unfractionated cellular extracts,El-dependent viralDNA replication is origin-dependent, but atelevated El concentrations, replication can occur on non-origin-containing DNA templates. This conversion from anorigin-dependent replication system to a nonspecific initiatorsystem is discussed in the context of the current understandingof the initiation of chromosomal DNA replication.

Helicases are critical enzymes in the semiconservative rep-lication of DNA (1). Although some polymerases can meltduplex DNA and progressively catalyze strand displacementahead of the growing polynucleotide chain, this melting isusually inefficient and requires the aid of a helicase. Heli-cases also work in conjunction with proteins having a strongaffinity for single-stranded (ss) DNA, which stabilize themelted duplex as the helicase catalyzes processive unwind-ing. Often a helicase is brought to the site on DNA wherereplication initiates by interaction with specific DNA bindingproteins that preassemble at the origin of DNA replication.For Escherichia coli or its A phages, the DnaB helicase isefficiently loaded onto the respective origin sites by DnaAand DnaC (2) or the A-encoded 0 and P proteins (3). Ineukaryotes, little is known as to how cellular helicases maybecome associated with replication complexes, although, forsome of the well-studied animal viruses such as the poly-omaviruses and the herpesviruses, viral-encoded helicasesare also equipped to be site-specific DNA binding proteinsthat can recognize the start site for replication. Other auxil-iary factors may, therefore, not be required for loading. Thesimian virus 40 large tumor antigen is such an origin-recognizing helicase (4) that initiates replication by unwind-ing the origin site as the complex cellular polymerizingmachinery assembles (5, 6). Herpes simplex virus 1 (HSV-1)encodes two proteins that can catalyze DNA strand displace-ment on helicase substrates, and one of these proteins bindsspecifically to the duplex HSV-1 origin of replication, al-though extensive duplex unwinding has not been detected (7,8).We have described an in vitro replication system for bovine

papilloma virus 1 (BPV-1) DNA (9, 10) that may provide

The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. §1734 solely to indicate this fact.

additional insight into the interactions between helicases andancillary replication proteins. For efficient replication, thiscell-free system requires two virally encoded proteins, Eland E2, and host cell extracts. A cis-acting origin of DNAreplication has been mapped, and the cell-free cis and transrequirements are similar to those for viral DNA replicationwithin the living cell (11, 12). Important differences do existbetween the two DNA replication assays. In the cell, the E2protein and DNA sequences that make specific contacts withE2 are absolutely required, whereas in the biochemical assayEl alone is sufficient and replication is enhanced by the E2protein, even in the absence of an E2 site. Considerableevidence supports the hypothesis that E2 stimulates replica-tion in part by aiding the El protein to bind viral DNA (refs.9, 10, and 13 and A. Stenlund, personal communication). Thiscooperativity is believed to be a function of protein-proteinand protein-DNA interactions (10). Thus, at sufficiently highEl concentrations, with naked DNA as a template for rep-lication, El binding may not be absolutely rate determining,and in vitro replication can be qualitatively independent ofE2. El, a 68-kDa phosphoprotein, is known to be a site-specific DNA binding protein (9, 10, 12, 14) that binds andhydrolyzes ATP (15, 16). We therefore asked whether El hashelicase and duplex unwinding activities. Here we show thatthe protein is an ATP-dependent helicase, but in the absenceof other factors, it can unwind circular duplex DNA promis-cuously in an origin-sequence-independent manner. Further-more, at high levels of El, origin-independent DNA replica-tion can be detected in the cell-free system.

MATERIALS AND METHODSDNA Templates. pLYSX (or SX; 5433 bp) was constructed

by inserting the Xba I-Sma I (nt 6132-945) origin-containingfragment of BPV-1 into pUC19. This fragment contains theentire upstream regulatory region. pLYIII (5170 bp) containsthe late-region BamHI-HindIII (nt 4450-6958) fragment ofBPV-1. pKS and pKSO were as described (9).DNA Helicase Assays. A standard DNA unwinding reaction

mixture (20 ,ul) contains 30mM Hepes (pH 7.5), 7mM MgC92,45 mM potassium glutamate, 4 mM ATP, 100-200 ng ofrelaxed circular DNA (3-6 x 10-14 mol), 40mM phosphocre-atine, creatine phosphokinase (100 ,ug/ml), bovine serumalbumin (50 ,ug/ml), E. coli single-stranded DNA bindingprotein (SSB; Pharmacia; 30 ug/ml), polyglutamic acid (150,ug/ml), and 10 units ofHeLa topo I. Additional experimentsshowed that removal of polyglutamic acid from the reactionmixture did not alter the properties of El reported. Reactionmixtures were titrated in the range of 1.4 x 10-12 to 7.4 x10-12 mol (100-500 ng) of El. After incubation at 32°C for 2h, reactions were terminated by adding 20 ,ul of stop solution,

Abbreviations: BPV-1, bovine papilloma virus 1; topo, topoisomer-ase; 2D, two dimensional; ss, single stranded; SSB, ssDNA bindingprotein; RPA, replication protein A; WT, wild type.*Present address: American Cyanamid Company, Lederle Labs, 401North Middletown Road, Pearl River, NY 10965-1299.tTo whom reprint requests should be addressed.

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to reach final concentrations of 1% SDS, 12 mM EDTA, andproteinase K at 60 ,ug/ml and then incubated for 30 min. Themixtures were extracted with phenol and chloroform. TheDNA products were fractionated in a 0.8% agarose gel andanalyzed by Southern blot hybridization. The strand-displacement assay was performed as described by Mohr etal. (17). A 42-base oligonucleotide was labeled by a kinasereaction and annealed to an M13 ss template, and thesubstrate was purified by Sephadex chromatography. Thereaction mixtures contained El as indicated and =10 ng oflabeled substrate, 30 mM Hepes (pH 8.0), 4 mM ATP, 7 mMMgCl2, and 0.5 mM dithiothreitol and were incubated at 37°Cfor 1 h. The reactions were terminated as described (17). Theproducts were then analyzed by PAGE in an 8% gel andautoradiography. For the helicase assay utilizing small du-plex DNA, the oril and ori- fragments were prepared bydigesting pKSO with HindIII, labeling the DNA with all four[32P]dNTPs and the Klenow fragment ofDNA polymerase I,and then digesting the mixture of DNAs with Pvu II. Thisgenerated a 406-bp ori+ fragment and a 258-bp ori- DNAfragment. These two fragments were purified by PAGE andused as substrates. The reaction conditions were identical tothose used in the unwinding assay, except that the topoisom-erase (topo 1) was omitted and that the total DNA substrateswere estimated to be 0.5 ng.

Replication and Two-Dimensional (2D) Gel Assay. In vitroDNA replication assays were done with modifications ofpublished conditions (9). Each reaction mixture (25 1,u) con-tains 30 mM Hepes (pH 7.5), 7 mM MgCl2, 45 mM potassiumglutamate, 4 mM ATP, 200 ng of DNA, 40 mM phosphocre-atine, creatine phosphokinase (100 ,g/ml), polyglutamic acid(150 ,g/ml), 10 ,ul of cell extract (8 mg/ml), and El andDNA-affinity-purified E2 as indicated. Other components areas described (9) or in the text. Reactions were incubated at37°C for 2 h and DNA was processed as described (9). Toanalyze replication intermediates, a 120-,ul reaction mixturewas assembled, scaling up the components from the standardreaction. Six samples were taken at various times through a2-h reaction and then pooled before analysis. For the origin-specific template, 500 ng of El and 60 ng of E2 were in themixture, and for the ori- reaction, 3 ug of El and no E2 wasemployed. For 2D gel analysis, 8 ,ul of DNA replicationproducts was digested with corresponding restriction en-zymes and then used for the analysis as described (18).

Electron Microscopy. Unwinding reactions using pLYSXas a template were incubated for 2 h at 32°C and then placedon ice. The DNA concentration was adjusted to 0.64 ,g/mlwith 10 mM MgCl2 and 10 mM Hepes (pH 7.5), and cy-tochrome c was added to a final concentration of 10 ,g/ml.Samples were spread by the aqueous drop method (19).

RESULTSEl Is a Helicase. To determine whether the El protein has

intrinsic helicase activity, the wild-type (WT) protein and amutant form (PS434) were expressed from baculovirus vec-tors and purified to homogeneity (>95%) as described (9).The PS434 allele with a Pro -* Ser codon change renders Elincapable of binding ATP and is defective for in vivo repli-cation (15). A helicase substrate was made by annealing alabeled oligonucleotide to a ssDNA circle (17). As shown inFig. 1A and B, the WT El was capable of displacing the linearDNA from the circle. However, the mutant PS434 had only10-20% of the WT activity. As anticipated, the PS434 mutantwas defective for in vitro DNA replication (data not shown).This helicase activity was dependent on ATP at all concen-trations of El tested (Fig. 1 C and D).El Can Initiate Unwinding from Within Duplex DNA. A

helicase assay more pertinent to substrates involved in DNAreplication uses topologically constrained duplex DNA. Co-

valently closed circular DNA was relaxed with topo I andthen incubated with El, ATP, SSB, and topo I. A highlyunwound (melted) duplex structure called form U can becreated by a helicase under such conditions, and after ex-traction of all proteins, the original population shows newsupercoiled forms when analyzed by agarose gel electropho-resis. Fig. 2A indicates that WT El could create such formson both ori+ DNA and an Ori- DNA template of similar size.Again, the mutant PS434 was defective in this assay. Noquantitative difference in apparent form U with SX (Ori+) orLYIII (Ori-) DNA was detected at any given level of Eltested. Furthermore, titration ofDNA templates from 0.1 to1.0 pig of DNA with constant El (500 ng) showed no quan-titative difference between Ori+ and Ori- DNA. A similarlack of origin specificity for unwinding in this type of assayhas been demonstrated for simian virus 40 large tumorantigen by Wold et al. (20). However, for tumor antigen, thenonspecific reaction was very sensitive to salt concentration.Presumably, the higher salt concentration increased therequirement for stronger DNA-protein interactions. At var-ious levels of NaCl, Oril and Ori- DNA were equivalentsubstrates for the unwinding catalyzed by El (data notshown). Fig. 2 shows that neither E. coli SSB nor humanreplication protein A (RPA; also an SSB) could create theunwound DNA species. However, since El at 500 ng withoutan SSB could produce the U form, El itself probably had highaffinity for ssDNA (Fig. 2, compare lane 3 with lanes 4-6).In other experiments (not shown here), we observed thataddition ofmammalian RPA to the reaction did not change oreliminate the Ori-independent nature of unwinding. Thereaction needed a nucleotide, but this requirement could besupplied by all four NTPs or dATP. The poorly hydrolyzableadenosine 5'-[y-thio]triphosphate was not an effective sub-stitute (Fig. 2C). The Km for ATP hydrolysis was 200 ,uM;therefore, at lower nucleotide concentrations, preferencesfor a particular nucleotide may indeed be observed.The assay described above indicates that a highly unwound

species can be created by El, but an extensive toroidal

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.FIG. 1. El is a DNA helicase. (A) El has DNA helicase activity.

The upper band is the starting substrate. The lower band representsthe displaced oligonucleotide as a result of helicase activity or boilingof the substrates. The amount of El protein (WT or mutant PS434,in ng) used in the reactions is indicated at the top of the figure. (B)Quantitation of displaced oligonucleotide shown in A. The intensityof each band was measured by PhosphorImager (Molecular Dynam-ics, Sunnyvale, CA). (C) ATP is required for the strand-displacementactivity. The helicase assay was carried out as described aboveexcept that ATP was omitted where indicated. (D) Quantitation ofthe helicase activity shown in C.

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A SX(Ori1-) LYIII(Ori-)WT PS434 WT PS434

M ---MEl (nCg) -C- 7N ° N LOD 8 CN4 NC LN O NUN

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FIG. 2. El can initiate unwinding from within duplex DNA. (A)Unwinding activity of WT El vs. mutant PS434 El. In each set oftitrations, El protein concentrations are indicated in ng. M, DNAmarker. Lanes: 2 and 12, no El was added. (B) Effects of SSB andRPA on unwinding. Lanes: 4 and 7, 600 ng of E. coli SSB; 5, 6, and8, 300, 600, and 600 ng of RPA, respectively; 2, 7, and 8, no El. (C)Effects of NTPs on unwinding. Lanes: 3, no NTPs; 2, 4-8, 4 mMATP, 4 mM CTP, 4 mM GTP, 4 mM UTP, 4 mM dATP, and 4 mMadenosine 5'-[y-thio]triphosphate (ATP-y-S), respectively; 1, no El.(D) Fragment unwinding assay. Equal molarity of an ori+ DNAfragment (406 bp) and an ori- fragment (258 bp) were mixed and usedas substrates. The lane labeled "boiled" provides a marker forssDNA positions. A band corresponding to the denatured ori+fragment is clearly visible, and one corresponding to the ori-fragment with 10%o of the intensity is detectable at the highestconcentrations of El.

wrapping of the DNA around El would also generate asupercoiled molecule with rapid gel mobility. To ascertainthat the faster-migrating species created by El did indeedrepresent unwound molecules, the reaction products wereexamined by electron microscopy. Under these conditions,the protein was not extracted from the templates, so simple6-shaped molecules were observed. As shown in Fig. 3,images characteristic of unwound molecules were detected.In the complete reaction mixture, we scored 107 molecules,23 (21%) of which were unwound; in the absence ofATP, 100molecules were scored and 1 (1%) was found with an appar-ent bubble.

In several assays, El can bind ori+ DNA in a sequence-dependent manner. We were therefore surprised that itshowed no specificity in the unwinding experiments de-scribed above. We reasoned that El may bind to nonspecificDNA with some affinity, and thus with a plasmid-sizedsubstrate, the difference in loading El between ori+ and ori-DNA may be too small for one to observe in a form U assay.Alternatively, lack of specificity could be the result of aspecial sequence in plasmid LYIII that could easily beunwound (the late region of BPV-1 has several patches ofA-T-rich runs). We therefore asked whether specificity inunwinding could be observed in an assay using small restric-

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FIG. 3. Electron microscope images of circular DNA unwoundby the El protein. Six representative pictures ofunwound moleculesare shown. After unwinding reactions, the DNA-protein complexeswere stained with cytochrome c and examined as described. (Bar inF = 100 nm.) The cytochrome c concentration was such that in manyexamples a clear distinction between duplex DNA and ssDNA wasclear. For example, inD the duplex DNA is not coated with SSB (orperhaps El) and is thus thin, whereas the denatured loops are coatedand appear thick. In B the distinction is lost presumably because ofexcessive cytochrome c staining. Such images were not detected inthe spreads from samples not containing El.

tion fragments of the BPV-1 URR. As shown in Fig. 2D, a406-bp oril fragment was the preferred fragment denaturedby El. In this assay, El showed a 5- to 10-fold specificity forthe ori+ fragment, and at the concentration of El used, thereaction was dependent upon a ssDNA binding protein andATP (data not shown). E2 interfered with the activity of Elin this fragment unwinding assay and in the form U experi-ments. Clearly, this effect requires further exploration, as ithas important implications for the fate of E2 after initiation.The OrIgn Requirement for in Vitro Replication Is El-Level-

Dependent. It is difficult to compare the specific activity ofElin a purified system to that of El in a crude extract. Forexample, the protein may bind nonspecifically to other pro-teins or be activated by specific associations with cofactors.Nevertheless, the above results, which showed that, in theabsence of competitor DNA, El could effectively unwindOri- plasmid DNA, led us to reexamine the absolute originsequence requirements for in vitro BPV-1 DNA replication;it is known that unwound nonspecific DNA can effectivelyserve as a template for most polymerase/primase systems(21). In Fig. 4, such a titration of El with Ori+ or Ori-templates for in vitro replication is shown. With 85 ng of El,almost no replication was detected on either template; how-ever, when supplemented with E2, a robust reaction wasobserved with Ori+ DNA as reported (9). With increasing Elconcentration, E2 had less effect. Significantly, with 500 ngof El, replication of pKS (Ori-) DNA was readily detected,albeit at much lower levels than was the Ori+ DNA. Incontrast to the specific replication, this BPV-1 Ori-independent replication was inhibited by E2 (Fig. 4, comparelanes 15 with 19).To ask whether these replication products represent true

de novo DNA synthesis or simply repair synthesis, Brewerand Fangman 2D gel analysis (18) was used. In this analysis,

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FIG. 4. Replication of Ori+ and Ori- templates in the cell-freesystem. (Upper) Titration of El protein for replication activity witheither Ori+ or Or- DNA templates. The amount of El added isindicated. E2 (20 ng) was added to the reaction mixtures whoseproducts are shown in the lanes labeled +E2. Thus lane 3 representsthe low level of replication detected with 85 ng of El in the reactionmixture and lane 7 shows the products with 85 ng of El and 20 ng ofE2. Replication products were isolated and fractionated by agarosegel electrophoresis. An autoradiogram is shown. (Lower) The abso-lute amount of dNTP incorporation was measured by scintillationcounting and converted to pmol of synthesis.

various families ofreplication intermediates were detected bycharacteristic arcs; such arcs cannot be generated by simplegap-filling mechanism or nick translation. When replicated,pKSO DNA was cleaved with Asp700 and analyzed; a bubblearc indicative of 0 forms (labeled B in Fig. SA) was detectedamong the replication intermediates. Asp700 cleaves thetemplate plasmid roughly 1800 from the specific origin site atan Hpa I site, and therefore, a family of simple bubbleintermediates is predicted. A family of simple Y forms wasalso predominant in this and in all 2D gels analyzed, probablybecause of nuclease activity in the extract. We also note thata conspicuous recombination pattern (labeled RI) was alsopresent in these patterns. As anticipated, when the replica-tion intermediates of pKSO (Ori+) were digested with Hpa I(which cleaves at the center of the origin), a double Y arc wasdetected and the bubble arc was lost (Fig. 5B). The productssynthesized on the Ori- DNA showed that the late double Yarc and the bubble arc were simultaneously detected, irre-spective ofwhat enzyme cleaved the DNA. Fig. 5C shows the2D gel pattern of the replication products of pKS digestedwith EcoRI; an identical pattern was obtained when digestedwith Asp700 (data not shown). These results show thatinitiation on Ori- templates does occur and that simple repairsynthesis does not account for the replication.

DISCUSSIONThe El protein ofBPV-1 is an ATP-dependent DNA helicase,and a point mutation in the ATP binding domain ofthe proteinrenders the activity defective. As El is a site-specific DNAbinding protein that binds to the origin, it seems reasonableto propose that the unwinding reaction plays a critical role inthe initiation reaction. In preliminary data on El helicaseactivity (9), we relied primarily upon strand displacement

Av

pKSO Hpa I pKS Eco RI

FIG. 5. 2D gel analysis of DNA products from the in vitroreplication. In vitro DNA replication and the 2D gel analysis wereperformed. (A) Replication products of pKSO were digested withAsp700. (B) pKSO was digested with Hpa I. (C) pKS was digestedwith EcoRI. When pKSO was used as a template, both El and E2were added to the reaction mixtures. When pKS was the template,El was the only viral protein included. Autoradiograms are shown.The patterns labeled B include the family of replication intermediateswith an internal bubble, where the bubble grows from the centerstoward the ends. The Y locates the family of intermediates with a Yshape where the fork grows toward one end. Similarly, the YYdenotes the family of double Y forms. RI locates putative recombi-nation intermediates (21). The location of the origin in the pKSOplasmid is shown below the autoradiogram by an open circle.

assays, which are less sensitive to low levels of contaminat-ing nuclease activity than are the unwinding reactions usingcircular DNA. Improved yields of El allow us to perform theassays on covalently closed circles. We have not been able tofind conditions in which the unwinding reaction on topolog-ically constrained DNA is absolutely dependent upon origin-specific sequences. Various titrations including substrateDNA concentrations, salt conditions, and protein levels didnot alter the specificity of the reaction. Unanswered ques-tions relate to the ratio of specific to nonspecific DNAbinding constants for El. In particular, we do not know howbroad a range of duplex sequences El can bind to and whatDNA sequence parameters determine the ease with which theprotein can initiate unwinding. Under stringent conditions ofDNase protein protection assays (9, 10, 12) or in a DNA-protein immunoprecipitation assay (14), El binds in a site-specific manner. However, in assays utilizing glutaraldehydefixation, El can be cross-linked nonspecifically to ori- DNA(unpublished observations). These results suggest that forEl-mediated unwinding in the absence of cofactors (or in-hibitors), it is sufficient for the protein to make relativelynonspecific contacts with the template.Given these concerns about the ability ofEl to bind duplex

DNA nonspecificaily, we asked whether the addition ofcompetitor DNA could shift the specificity of unwindingcircles toward origin-containing DNA. In titrations of suchcompetitor A DNA, even in a 10-fold excess of carrier, littledifference was observed between pLYIII (ori-) and pSX(ori+) DNA (data not shown). Nevertheless, we believe thatin the in vitro replication reactions, El is involved in un-winding circularDNA in an origin-sequence-dependent man-ner. Significantly, utilizing small DNA fragments, El showspreference for melting the origin-containing fragment. Theseexperiments show that under certain conditions El caninitiate unwinding from within duplex DNA in a site-directedmanner. These fragment assays are different from the form U

A B C

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assay and are independent of topos. Moreover, if the size ofa specific binding site is roughly equivalent to the size of anonspecific site, the specific to nonspecific site ratio isgreater in the fragment unwinding assay than in the U formassay. Clearly, it will be important to understand whatsequences, if any in particular, are required for El-mediatedunwinding of pLYIII (oril) DNA. The fragment assay does,however, indicate that El may be an effective initiator if thenonspecific reaction is reduced or if El can be made to bindmore specifically. E2 may play some role in this regardalthough we point out that in the cell-free replication system,El alone at protein levels identical to those used in the formU assays can lead to origin-specific replication. Ultimately,the proper template that most mimics in vivo conditions forthe study of BPV-1 DNA replication is a chromatin complex.The core histones, when associated with the template, mayeffectively lower the concentration of nonspecific DNAsequences in the late region of the viral genome and allow fora more specific assembly of the replication apparatus at theorigin.The in vitro DNA replication system shows similar spec-

ificity for the cis and trans components expected fromexperiments performed within the cell. Thus, over a broadrange of El concentrations, it is origin-dependent and re-sponds to E2. The lack of specificity in the unwinding assayled us to ask whether we could find sufficiently high con-centrations of El to promote origin-independent replication.Although this origin-independent replication is 5-fold lowerthan that detected with ori+ DNA, the 2D gel analysissuggests that the synthesis is replicative and not repair.Under origin-dependent conditions, previous measurementsof incorporation of radiolabel into restriction fragmentsshowed that the bulk of the actual initiation in the cell-freereplication system occurs near the origin. Thus we attributethe predominant Y arcs in Fig. 5 A and B to the breakage ofthe intermediates by shear force or nuclease activity. How-ever, we cannot exclude the possibility that even under lowEl concentrations, where the origin sequence is required,some initiation occurs outside the origin site.

Further study of the origin-sequence-independent replica-tion is warranted for it may be relevant to in vivo replicationof nonviral DNA in a variety of systems. Although specificregions of initiation for DNA replication have been defined atcertain loci in mammalian cells, it is only in budding yeastwhere definitive evidence shows a genetic requirement forspecific origin sequences (21). It is conceivable that replica-tion occurs reproducibly at certain loci without a true geneticrequirement for those specific sequences. In studies ofDNAreplication in Xenopus laevis eggs (22, 23), Parameciumtetraurelia (24), nematodes (25), and human cells (26), itappears that transforming DNA can replicate and be main-tained without a strict requirement for sequence. The per-missive state of these systems may reflect an accessibility ofthe DNA template to an abundant helicase that can unwindDNA with a relaxed sequence specificity. Such helicases mayhave affinity for the polymerase a-primase complex. Teth-ering the polymerase to such an unwound locus may lead toan initiation site. We know that El does have such affinity forthis polymerase (unpublished data), and the switch from anorigin-dependent system to an independent one in the cell-free system seems to be a function of El levels. In this regardit is intriguing that Burnett et al. (27) identified by electronmicroscopy a large number ofapparently randomly dispersedeye forms in the BPV-1 DNA extracted from bovine lesions.Though these eye forms were not shown by direct methodsto be replication initiation sites, it was suggested that BPV-1replicates at this stage in the viral life cycle from a multitudeof start sites. It is possible that in vegetative BPV-1 replica-tion, El levels are considerably higher than those reached in

latently infected cells, where a unique origin sequence ap-pears necessary for plasmid replication. Furthermore, it willbe important to learn whether overexpression of El fromrecombinant vectors can lead to a relaxed requirement forspecific origin sequences in vivo. Under such conditions, lowlevels of replication may start from other sites (or severelydivergent ori sequences) in the viral genome.

Finally, we mention that the enzymatic activity of Elreported here may be helpful in a variety of clinical studies.The El genes from other papillomaviruses, including those ofhuman origin, can substitute for the BPV-1 El product in thecell for viral DNA replication (refs. 28 and 29 and J. Russelland M.B., unpublished data). It thus seems likely that allpapillomaviral El genes encode an ATP-dependent helicasewith properties equivalent to the BPV-1 product. We knowthat the human papillomavirus 6b El product is a site-specificDNA binding protein with ATPase activity (L.Y., J. Russell,R. Li, and M.B., unpublished data). Therefore, a single drugthat inhibits this helicase activity may serve as an effectiveuniversal agent against these human pathogens.

We thank Drs. B. Stillman and T. Kelly for generous gifts of thehuman RPA and Drs. N. Cozzarelli, R. Kanaar, and J. Hurwitz forstimulating conversations. This work was supported by grants fromthe U.S. Public Health Service (CA42414 and 30490).

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