Biochemical and Electron Microscopic ImageAnalysis of the Hexameric E1 Helicase*

(Received for publication, September 28, 1998, and in revised form, December 2, 1998)

Erik T. Fouts‡§, Xiong Yu¶, Edward H. Egelman¶, and Michael R. Botchan‡i

From the ‡Department of Molecular and Cell Biology, University of California, Berkeley, California 94720 and the¶Department of Cell Biology and Neuroanatomy, University of Minnesota Medical School, Minneapolis, Minnesota 55455

DNA replication initiator proteins bind site specifi-cally to origin sites and in most cases participate in theearly steps of unwinding the duplex. The papillomaviruspreinitiation complex that assembles on the origin ofreplication is composed of proteins E1 and the activatorprotein E2. E2 is an ancillary factor that increases theaffinity of E1 for the ori site through cooperative bind-ing. Here we show that duplex DNA affects E1 (in theabsence of E2) to assemble into an active hexamericstructure. As a 10-base oligonucleotide can also inducethis oligomerization, it seems likely that DNA bindingallosterically induces a conformation that enhanceshexamers. E1 assembles as a bi-lobed, presumably dou-ble hexameric structure on duplex DNA and can initiatebi-directional unwinding from an ori site. The DNAtakes an apparent straight path through the double hex-amers. Image analysis of E1 hexameric rings shows thatthe structures are heterogeneous and have either a 6- or3-fold symmetry. The rings are about 40–50 Å thick and125 Å in diameter. The density of the central cavityappears to be a variable and we speculate that a pluggedcenter may represent a conformational flexibility of asubdomain of the monomer, to date unreported forother hexameric helicases.

The synthesis of duplex DNA is a complex enzymatic processthat requires the coordination of large numbers of proteins.The mechanisms are elaborate in part because the enzymesthat use the complementary template strand as a guide fornucleotide incorporation catalyze this synthesis only in the 59to 39 direction. Given the antiparallel nature of the duplex thisusually requires that two synthetic enzymes move in oppositepolarities on the two strands. Synthesis of the so-called laggingstrand is discontinuous and requires the cyclical association ofthe enzyme, while synthesis of the other strand is continuous.Nevertheless, in many prokaryote replication systems it isclear that coordination of these enzymes is achieved and main-tained by a dimeric polymerase that creates a looped DNAstructure in the lagging strand. This loop is mediated by mul-tiple protein-protein interactions across the growing fork (Ref.1, and references therein). Helicases are enzymes that cancatalyze the unwinding of the template strands ahead of thefork, thus allowing for new complementary strand DNA syn-

thesis. They were initially discovered as ancillary factors re-quired for synthesis, but recently this view of the helicaseactivity has been characterized as “naive” or at least incom-plete (2). Compelling evidence has been presented demonstrat-ing that the helicase is an integral member of a large proteincomplex that serves as a molecular motor or pump for thereplication apparatus empowering the polymerase and increas-ing the rate of DNA polymerase synthesis (3). The Escherichiacoli dnaB helicase also plays a critical function in establishingthe asymmetry at the growing fork. The helicase tracks on thelagging strand template but through interactions it holds theleading strand DNA polymerase while allowing for recycling ofthe other DNA polymerase (4). How helicases actually convertthe binding and hydrolysis of ATP into mechanical energyresulting in DNA unwinding and can concomitantly achieverelative movement along the DNA is presently under intenseinvestigation (5, 6). While many issues remain unresolved, itseems as if the well studied replication helicases of E. coli (andits phage encoded ones) engage DNA by encircling at least oneof the DNA strands that have been prepared for this loading byother replication proteins (7–10). Thus, for example, the dnaBhelicase is loaded onto DNA in complex with dnaC to a duplexstructure at oriC already melted by the dnaA protein (11).

In eukaryotes, despite the ubiquitous presence of many DNAhelicases (12), little is known about the relationship betweensuch enzymes and the replication complex. However, the im-portance of such proteins in eukaryotic DNA replication ishighlighted by the fact that many DNA viruses that replicate inthe nucleus encode a helicase. One type of such viral helicasecan initiate unwinding from within duplex structures not pre-pared for activity by prior melting. These helicases encoded bythe herpes simplex virus, papillomaviruses or the SV40, andpolyoma viruses can serve as DNA initiators by first recogniz-ing small repeat motifs within the origin of replication. Thus, aparticularly challenging structural problem exists in determin-ing how these proteins convert from a site-specific DNA bind-ing mode to a helicase. For the SV40 T antigen, monomers bindto pentameric base pair repeats utilizing specific nucleotidebase information. After double hexamer formation on the DNAand ATP binding DNA-protein contacts shift toward sugar-phosphate interactions (13). A complex series of steps musttherefore occur to change both the oligomeric state of the pro-tein and the nature of its contacts with DNA. Presumably, boththe DNA and ATP could be allosteric effectors of this change,but in the case of T antigen ATP is sufficient for hexamerformation. Similarly for the HSV-1 origin binding protein UL9a pair of dimers interact with each other and bend the oriregion as duplex DNA-binding proteins. In the presence of ATPthe complex becomes an active unwinding enzyme that canextrude catenated single-stranded loops (14).

The papillomaviruses provide a unique system for analyzingthis assembly and transition process. The bovine papilloma

* This work was supported by National Institutes of Health GrantsCA42414 and CA30490 (to M. B.) and GM35269 (to E. H. E.). The costsof publication of this article were defrayed in part by the payment ofpage charges. This article must therefore be hereby marked “advertise-ment” in accordance with 18 U.S.C. Section 1734 solely to indicate thisfact.

§ Present address: Life Science Div., Lawrence Berkeley NationalLaboratory, Berkeley, CA 94720.

i To whom correspondence should be addressed. Fax: 510-643-6334;E-mail: mbotchan@uclink4.berkeley.edu.

THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 274, No. 7, Issue of February 12, pp. 4447–4458, 1999Printed in U.S.A.

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virus type 1 (BPV-1)1 encodes a 68-kDa phosphoprotein (E1)that binds site specifically to two sets of short repeats orga-nized as inverted repeats at the viral origin of replication (15,16). The protein is also an ATPase (17) and a helicase able toinitiate unwinding from within a duplex DNA circle (18, 19).However, unlike UL9 or the SV40 T antigen, E1 requires invivo an ancillary viral factor in order to be targeted to the viralorigin of replication (20, 21). The E1 protein in a cell-free DNAreplication system can direct origin-specific DNA replication;however, this activity is greatly stimulated by E2 and at lim-iting dilutions of E1 the in vitro DNA replication becomesabsolutely dependent upon E2 (22). This dependence upon E2reflects the cooperative interaction between E1 and E2 in bind-ing to viral DNA (22–25). The E2zE1zDNA complex initiallyassembled at the origin consists of two or four monomers of E1spanning the inverted repeats and an E2 dimer occupying anadjacent binding site (15). Interestingly, the E2 dimer mustleave this complex in an ATP-dependent reaction before high-er-order complexes of E1 assemble (26, 27). Therefore, it seemsas if E2 might stabilize the site-specific DNA binding confor-mation of E1 and either mask or allosterically block the heli-case transition. In vivo, this chaperone process seems likely tohave evolved to increase the likelihood of origin occupancy andperhaps to allow for the coordination of other activities of E1with the transcriptional and segregation functions of E2 (28,29). In any case understanding the differences between the E1assembled in the preinitiation E1zE2 complex and as an activehelicase on the DNA should provide insights into the transi-tions that must occur for all of the viral initiator proteinsdiscussed above.

E1 is believed to be structurally related to the SV40/polyomavirus-encoded large T antigens. Both helicases track on theleading strand template (23), and the overall organization ofthe open reading frames are similar. For instance, the nuclearlocalization domain is proximal to the site-specific DNA-bind-ing domain and the conserved “Walker” A and B boxes of theATP-binding domains are equivalently spaced and are about200 amino acids displaced from the DNA-binding domain (30).Moreover, mutations of these conserved motifs affect activity inexpected ways (17, 19, 31, 32). However, many of these align-ments can now be made for other members of the SF3 family ofDNA helicases that do not have analogous activities such asthose from parvoviruses and the human herpes 6 virus (33). Itis therefore important to analyze how E1 as an active unwind-ing enzyme actually engages DNA. In this report we use bothbiochemical and electron microscopy techniques to establishthat E1 initiates unwinding from the origin site and unwindsDNA bidirectionally; moreover a bi-lobed double hexamericcomplex similar to the images obtained for T antigen wasobserved at the start site. In these complexes the DNA is likelyto take a straight path through the double rings. Biochemicaland electron microscopic analysis showed that, as anticipated,unwinding activity correlates with the formation of hexamers.Image analysis of the hexameric structures showed that themolecules do form toroidal rings with a central hole; thesemolecules possess either 3- or 6-fold symmetries. Surprisingly,a significant fraction of the hexamers show density in thecentral hole. Such “filled” centers have not been observed forother hexameric helicases but we speculate that the proteinhas several conformational states and that conformational flex-ibility may indeed be a general feature of this family.

MATERIALS AND METHODS

Plasmid Construction and DNA Substrates—pKSO has been de-scribed previously (22). pSS3, pSS3-LI5C, and pSS3-Dopal are de-scribed by Mendoza et al. (16). The BPV-1 origin containing fragmentgenerated by BamHI and HindIII restriction enzyme digest of pKSOwas inserted into the pACYC177 vector linearized by BamHI and Hin-dIII to give rise to pCLO. The 429-base pair BPV-1 origin containingsubstrate DNA used in the fragment unwinding assay and for DNAinduced E1 oligomerization was generated by digesting pKSO with theEcoRI and PvuII restriction endonucleases. The 242-base pair origincontaining DNA fragment used in EM linear compaction studies wasgenerated by digesting pKSO with EcoRI and BamHI restriction endo-nucleases. Both of the duplex fragments were purified from agarosegels. The sequence of the 10-base pair oligomer used for DNA inducedE1 oligomerization is 59-AACAACAATC-39. The E1 construct used foroverexpression in E. coli, pGEX-2TK-E1, was generated by cloning theE1 open reading frame from the pET11-GST-E1 plasmid (25) into thepGEX-2TK vector (Pharmacia number 27-4587-01, GenBank accessionnumber U13851). The integrity of the boundaries for the E1 codingsequence was verified by DNA sequencing.

Protein Purification—The BPV-1 E1 protein was purified from Sf9cells infected with a recombinant baculovirus expression vector byimmunoaffinity chromatography as described by Yang et al. (22). TheE1 protein purified from E. coli began with transforming XA90 cellswith the pGEX-2TK-E1 expression vector and proceeded according tomethods described by Sedman et al. (25) as modified by C. Sanders.2 Inbrief, extracts from isopropyl-thio-b-D-galactopyranoside-induced cellswere prepared, cleared of nucleic acid by a Polymin P (10% w/v) pre-cipitation (0.5% w/v final) centrifuged by a 25,000 3 g spin for 20 min.E1 protein in the supernatants was precipitated by 65% ammoniumsulfate at 4° C. The recovered protein was purified by adsorption toglutathione-Sepharose beads and eluted in buffer containing 20 nM

glutathione. The GST moiety was cleaved with thrombin and the E1was further purified by chromatography on an S-Sepharose column(Pharmacia). The E1 containing fractions were pooled, dialyzed againstE1 dialysis buffer (20 mM pKPO4, pH 7.5), 150 mM potassium gluta-mate, 1 mM EDTA, 1 mM DTT, and 10% glycerol aliquoted, and storedat 280° C.

Unwinding Assays—The unwinding reactions using either co-valently closed circular DNA or duplex DNA fragment substrates wereperformed as described previously (19).

Electron Microscopy and Measurement of DNA Regions—Unwindingreactions using substrates indicated in the text were incubated at 32° Cfor 1 h. Micrographs of linearized DNA were prepared by adding 6 unitsof the indicated restriction enzyme and incubating for an additional 20min. Reaction products were fixed by the addition of glutaraldehyde to0.6% and purified by filtration through a 0.5-ml Bio-Gel A5-M column(Bio-Rad) and applied to glow-discharged carbon grids coated with 2 mM

spermidine. The grids were then rotary shadowed with tungsten. Pho-tographs were taken at 3 30,000 with a JEOL 1200 EX electron micro-scope at an acceleration voltage of 80 kV (61). Micrographs of E1complexes bound to a BPV-1 origin containing DNA fragment for linearcompaction studies were prepared using the same method describedabove. Measurement of duplex regions was performed by projectingphotographic negatives onto a Numonics digitizing tablet.

Image Analysis—The E1 protein (3 mM concentration, in 25 mM

KPO4, 60 mM NaCl, 1 mM EDTA, 1 mM DTT, pH 7.5) was incubated with18 mM of a 60-mer oligonucleotide for 30 min at 30° C, and then appliedto glow-discharged grids and stained with 2% (w/v) uranyl acetate.Electron micrographs were recorded under minimal dose conditions(with no prior exposure to the high magnification electron beam prior torecording) at 3 30,000 magnification, using a JEOL 1200 EXII micro-scope. Negatives were scanned with a Leaf 45 microdensitometer, witha sampling interval of 4 Å/pixel. Images of rings were masked into 44 344 pixel arrays (corresponding to 176 3 176 Å), band-pass filtered(between 1/160 and 1/12 Å21), scaled to zero mean density, and thecontrast was normalized. After applying a reference-free alignment(44), images were ranked by the strength of either the 3- or 6-foldpower, as described in Yu et al. (39). For the 6-fold ranking, we excludedthose images that contained a significant 3-fold power. After sortingbased upon rotational symmetry, images were then sorted based uponthe strength of the integrated density within a 16-Å radius area at therotational axis of the ring.

Radiolabeling of E1—The 7 amino acid NH2-terminal tag on the E1purified from overexertion in E. coli contains a recognition sequence as

1 The abbreviations used are: BPV, bovine papiloma virus; GST,glutathione S-transferase; DTT, dithiothreitol; SSB, single stranded-binding protein; PAGE, polyacrylamide gel electrophoresis. 2 C. Sanders, personal communication.

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well as a serine that can be specifically phosphorylated by bovine heartmuscle kinase. A typical 30-ml labeling reaction contains 2.5 mg of E1(1.3 mM final), 10 units of bovine heart muscle kinase (Sigma P-2645),and 2 ml of [g-32P]ATP at 6000 Ci/mM in 20 mM Tris (pH 7.5), 100 mM

NaCl, 12 mM MgCl2, and 0.1 mM DTT. The reaction is incubated at 4° Cfor 30 min and stopped with the addition of EDTA to 25 mM. E1 waspurified away from free 32P and nucleotides by Sephadex G-25 columnchromatography (NAP-10 column, Pharmacia number 17-0854-02).

Electrophoresis of E1 Complexes—Native gel electrophoresis of E1was performed as indicated in the text. For analysis of cross-linked E1complexes, the E1 sample was boiled for 5 min in Laemmli SDS loadingbuffer (pH 8.8) 1 130 mM b-mercaptoethanol. The samples were thenloaded onto polyacrylamide gradient gels containing an acrylamide:bisratio of 80:1. A stacking phase was not used and electrophoresis wascarried out in 25 mM Tris, 250 mM glycine, 0.1% SDS with a final pH of8.8. Current was held constant at 10 mA.

Glycerol Gradient Sedimentation—E1 purified from Sf9 cells infectedwith a recombinant baculovirus was centrifuged in a 15–35% glycerolgradient with 0.1 KCl-HEMG 1 0.01% Nonidet P-40 (62). For E1purified from overexpression in E. coli, the protein was centrifuged asabove with the following modification: a gradient of 15–37% glycerol in1 M NaCl, 20 mM HEPES (pH 7.5), 5 mM EDTA, and 0.01% Nonidet P-40was used.

RESULTS

E1 Unwinds DNA Bidirectionally from the ori Site—BPV-1replicates bidirectionally in vivo and in cell-free extracts (22,34, 35). With purified replication components wherein E1 pro-vided the only helicase activity (36), fully replicated circleswere obtained. Furthermore, acting on covalent closed circles,E1 is capable of producing a highly unwound DNA (form U)consistent with complete denaturation of the circles (19, 23). Itwas therefore anticipated that E1 as a replicative helicasemust be capable of processive unwinding that spreads bidirec-tionally from the BPV-1 origin region. Electron microscopy andan in vitro unwinding assay were employed to map both thelocation and extent of unwinding of BPV-1 genomic DNA by E1.Covalently closed circular DNA was relaxed with calf thymustopoisomerase I and then incubated with purified E1, ATP,topoisomerase I, and E. coli SSB protein. For this analysis, weused the plasmid pSS3 that contains an intact BPV-1 genomecloned into pUC18. The samples were fixed with glutaralde-hyde, purified by gel filtration, and linearized by restrictionendonuclease hydrolysis at a unique site. Representative im-ages are shown in Fig. 1, A-D. The lengths of the unwoundregions of DNA and total contour lengths were measured todetermine both the size and position of the unwound regionswith respect to the entire length. A compilation of data ispresented in Fig. 1E. As each of the molecules could be alignedin either of two directions (and one such orientation chosen foreach to make the alignment) it was necessary to repeat thisanalysis with a different unique single cutter. This separate setincreases the statistical significance of the conclusions. Suchdata obtained with either AflII or SacI defining the ends (Fig.1E) show that unwinding initiates at the ori site and thatdenaturation spreads bidirectionally from that position.

These results are compatible with those presented previ-ously by Seo et al. (18), who showed that E1-dependent forma-tion of form U was dependent upon the integrity of ori. How-ever, our earlier results (19) on form U production showed littledependence upon origin sequences, a result that might predictscattered bubble positions. With different preparations of theE1 protein we performed in vitro unwinding assays with vari-ous mutant templates. Mutations were engineered into theE1-binding site to determine whether the E1 DNA-binding sitecontributed quantitatively to the level of form U DNA pro-duced. Two mutants were derived from the plasmid pSS3; LI5Ccontains a 5-base pair linker insertion between the invertedrepeat, and for D OPAL the entire palindrome is deleted. Thereaction products for each were separated by agarose gel elec-

trophoresis, blotted to filters, and probed with pUC18. Phos-phorImage analysis of these Southern blots was used to deter-mine the amount of form U DNA generated (Fig. 2). The datado show that while all substrates are capable of directingunwinding the wild type ori site is indeed preferred. It there-fore seems likely that when a template contains a bona fide oriwith E1 sites organized in such a way as to allow for helicaseformation, such sites will be utilized in vitro. We do not under-stand why such preferences were not detected earlier, butperhaps this specificity is sensitive to monomer E1 concentra-tion and variations in this regard might influence the data.

The E1 Protein Forms Hexamers and Oligomerization Cor-relates with Helicase Activity—To determine the oligomericstates of E1 we analyzed the sedimentation profiles of thebaculovirus-purified E1 incubated in the presence and absenceof ATP. Fractions from a glycerol gradient were collected, andthe positions of the E1 protein determined by SDS-PAGE andWestern blotting using polyclonal E1 antisera. The proteinprofiles of the gradients (Fig. 3) indicate that E1 purified bysingle step affinity chromatography sediments in a heteroge-neous manner. In addition, it is clear from this analysis thatATP is not sufficient for the oligomerization of E1 monomers.The molecular mass markers suggest that the slowest migrat-ing peak corresponds to monomeric E1 (68 kDa) and the secondpeak a hexameric form (408 kDa). Fractions at the bottom ofthe tube likely correspond to aggregates. Glycerol gradientfractions containing the putative hexamer fraction and mono-mer fractions were analyzed by electron microscopy. The im-ages of the putative hexamer peak showed a typical 6-mem-bered toroidal structure (Fig. 3) bearing striking similarity tothe published micrographs of hexameric helicases (8, 10, 37–39). The monomeric peak showed no such structures (data notshown).

Protein preparations from baculovirus vectors showed a mix-ture of forms, and to study a more homogeneous population andto investigate the relationship between the monomer and hex-amer forms in more detail, we purified E1 from E. coli cellsusing the methods described by Sedman et al. (25). Indicationsfrom spectrophotometric 280/260 absorption ratios for baculo-virus E1 preparations were that the yields of hexameric peaksand aggregated material correlated with trace nucleic acidcontaminations. We therefore explored the notion that DNAbinding might be a factor in oligomerization. The E. coli E1possesses an amino-terminal sequence which can be phospho-rylated to high specific activity in vitro; such modification hasno effect upon helicase activity or other biochemical tests de-scribed below (data not shown). The purified material sedi-mented as a homogeneous monomeric fraction (Fig. 4B). More-over, this protein’s sedimentation behavior was not affected byATP binding. The E. coli protein was found to be active incell-free DNA replication and its activity was stimulated by E2(data not shown).

The monomeric radiolabeled E1 was incubated with a 429-bpduplex DNA fragment in the absence of ATP and Mg21. Reac-tions were fixed employing titrations of glutaraldehyde andsubjected to denaturing polyacrylamide gradient electrophore-sis (Fig. 4A). A ladder of cross-linked phosphorylase or com-mercial prestained standards (Kaleidoscope, Bio-Rad) wereused as gel standards. The results show that duplex DNA canpromote oligomerization and the data confirm that hexamericforms of E1 predominate. In the absence of DNA no multimer-ization was detected at any concentration of cross-linkingagent. These data were obtained both with nonspecific duplexand single-stranded DNA. Even very small oligonucleotidescan catalyze this oligomerization. In the presence of a 10-basesingle strand oligomer (in the absence of ATP), E1 sediments as

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a hexamer in a glycerol gradient (Fig. 4B). Analysis of theprotein peaks collected from these gradients (after glutaralde-hyde fixation) by denaturing acrylamide gel electrophoresisconfirms that the high and low molecular weight peaks repre-

sent hexameric and monomeric E1 forms (Fig. 4C).To explore the functional significance of this oligomerization

we sought to correlate E1 unwinding activity with its multim-eric state. To obtain such correlations we chose the duplex

FIG. 1. Electron micrographs of un-wound BPV-1 origin containing plas-mid DNA. Unwound pSS3 DNA mole-cules cleaved with either AflII (A and B)or SacI (C and D) restriction enzymes.The molecules were applied to carbongrids and rotary shadowed with tungsten,as described under “Materials and Meth-ods.” The single-stranded DNA appearsthicker than duplex DNA, because thesingle-stranded DNA is coated with E.coli SSB and/or E1. Bar in D 5 200 nm. E,measurements of unwinding of BPV-1 or-igin containing DNA in vitro. To deter-mine the extent, direction, and location ofunwound regions, molecules were photo-graphed, and the length of duplex DNAwas measured by projecting negativesonto a Numonics digitizing tablet. The po-sitions of unwound regions (black boxes)were plotted below a linear map of theplasmid. The relative position of the ori-gin in base pairs is indicated at the bot-tom of the histograms. The standard ofdeviation for total length was 9%.

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unwinding assay utilizing a duplex restriction fragment thatcontains the BPV-1 ori sequence. In previous experiments fromour laboratory (19), unwinding in this assay was dependentupon ori sequences and absolutely required an SSB. In twoparallel sets of reactions we either followed the oligomeric stateof E1 across a range of protein concentrations (3.75 to 480 nM)(Fig. 5A) or the state of the duplex DNA (4.2 nM) over the sameprotein titration (Fig. 5B). In these side-by-side experimentsreaction conditions were identical with the exception that SSBwas not present in the data obtained for Fig. 5A. As E1 assem-bles on the DNA in the presence of ATP/Mg21 and E. coli SSB,the duplex is melted and converted to single strands. Theability of E1 to act as a duplex unwinding enzyme is verycooperative (Fig. 5C) with respect to concentration and corre-lates with oligomerization. Hexamers and high forms (perhapsdouble hexamers) correlate with such activity. At 60 nM E1some unwinding is first detected and at this concentrationhexamers and notably higher forms are first detected.

DNA Takes an Apparent Linear Path Through a Bilobed E1Complex—From the data in Fig. 5 there is a suggestion thatcomplexes of higher order than hexamer may be the mostefficient in unwinding duplex DNA. For SV40 T antigen directexperiments indicate that a double hexameric form of thishelicase may provide the most effective enzymatic complex forsuch purposes (40, 41). To extend this comparison we asked ifa bilobed structure, taken as a measure of double hexamerformation for T antigen (42, 43) could be observed for E1.Purified E1 protein was incubated (in the absence of ATP) witha 242-base pair duplex DNA fragment containing a centrallylocated ori sequence. Electron micrographs were recorded ofthe protein-DNA complexes (Fig. 6A). The characteristic “dou-ble doughnut” images were prevalent. The length of the DNAfragments with and without bound protein was measured byprojecting electron micrograph negatives onto a numonics dig-itizing tablet (Fig. 6B). We found that the DNA length distri-bution does not change upon engaging E1. This absence of alinear compaction of the protein-bound DNA indicates that theDNA likely takes on a linear path through the E1-proteincomplex. The data clearly rule out any mode of binding which

would require the DNA to wrap around the helicase (Fig. 6C).Similar DNA compaction studies performed on the E. coli dnaBhelicase (7) and T antigen (42, 43) have been similarly inter-preted; however, we would point out that the standard devia-tion in length measurements (;9%) is very close to what mightbe expected for more complex models wherein one strandpasses through one hexamer and out through the top of thesame shell (see line 2, Fig. 6C). To resolve this point higherresolution or other approaches to analyzing these structures isrequired.

Image Analysis of E1 Hexameric Ring—To obtain a cleareranalysis of the ring structures, minimal dose electron micros-copy and image analysis were used to study the organization ofthe oligomeric state of the E1 protein. Fig. 7 shows electronmicrographs where both top views of the rings (Fig. 7a, formedwith an oligonucleotide) and side views of the rings stacked ondouble-stranded DNA (Fig. 7B) can be seen. Images of 968 topviews of the rings were averaged together, using a reference-free alignment procedure (44). The resulting average (notshown) suggested a hexameric structure, but subsequent anal-ysis indicated that the population of rings was non-homoge-neous. First, a sorting by rotational power (as done for DnaBprotein in (39)) indicated that a subset of the rings had asignificant 3-fold rotational power, consistent with the asym-

FIG. 2. E1 helicase prefers origin-containing substrates. Un-winding reactions were performed with the following substrates: plas-mid pSS3, which has a wild-type E1-binding site, plasmid pSS3-Dopal,which lacks the E1-binding site, or plasmid pSS3-LI5C which has a5-base pair linker insertion between the inverted repeats that make upthe E1 DNA-binding site. The E1 concentrations were held constant(368 nM) and l genomic DNA was titrated into the reactions as nonspe-cific competitor DNA. The reaction products were Southern blotted andradioactivity quantitated by PhosphorImager analysis. The amount ofhighly unwound form U DNA generated by helicase activity is plotted.The graph indicates that substrates with wild-type E1-binding sites aremore effectively unwound than those lacking E1 sites.

FIG. 3. Glycerol gradient analysis of E1. 4.5 mg of purified E1 (1.3mM final concentration) was incubated for 10 min at 37° C in 20 mM

HEPES (pH 7.5), 50 mM KCl, 7 mM MgCl2, in the presence or absence of4 mM ATP. The reactions were subjected to centrifugation throughgradients of 15–35% glycerol as described under “Materials and Meth-ods.” Fractions were collected and subjected to SDS-PAGE and Westernblotting. A Western blot of all fractions across the gradients is shown.The position of molecular weight markers run in parallel gradients isindicated above each protein profile (669 kDa, thyroglobulin; 232 kDa,catalase; 66 kDa, bovine serum albumin). The starting material wasloaded in the lane following the last collected gradient fraction and islabeled LOAD. The protein profiles of the gradients indicate E1 sedi-ments as two species of approximately 68 and 400 kDa. The materialhaving a molecular mass greater than 669 kDa is thought to representhighly aggregated forms of E1. The glycerol gradient fraction contain-ing the high molecular mass species of E1 (in the presence of ATP,molecular mass approximately 400 kDa) was applied to glow-dis-charged carbon grids and stained with 2% (w/v) uranyl acetate. Theelectron micrographs shown were taken at 3 30,000 with a JEOL 1200EX electron microscope at an acceleration voltage of 80 kV. Bar, 250 Å.

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metric unit in these rings being a dimer. Trimers of dimers haspreviously been observed for the hexameric rings formed by theDnaB protein (39, 45) and the RecA protein (46). Fig. 8, a andb, show the 6-fold symmetric averages (containing 678 rings),while Fig. 8, c and d, show the 3-fold symmetric average (con-taining 100 rings). The rotational power spectra for the 6- and3-fold symmetric averages are shown in Fig. 8, i and j, respec-tively. The main difference between the 6-fold symmetric rings

and the 3-fold symmetric ones is that there appears to be amodulation of the projected subunit density in the 3-fold sym-metric averages, such that there are alternating “strong” and“weak” subunits. In addition, the outermost ends of the subunitarms appear to move in toward the center for the three weakersubunits in the 3-fold conformation. Both forms of the ringappear to be about 125 Å in diameter.

Second, the density within the central channel appeared to

FIG. 4. DNA stimulates E1 oligomer-ization. A, radiolabled monomeric E1protein (128 nM) was incubated in thepresence (lanes 6–10) or absence (lanes1–5) of a 429-base pair BPV-1 origin con-taining DNA fragment (3.8 nM). Glutaral-dehyde was titrated into the reactions(0.005, 0.010, 0.020, and 0.040% final) fol-lowed by boiling in SDS sample buffer 1b-mercaptoethanol. The reactions wereanalyzed by electrophoresis on a denatur-ing gradient gel (4.4–20% acrylamide)and stained with Coomassie BrilliantBlue to allow identification of molecularweight markers. This was followed bydrying of the gel and autoradiography. Anautoradiogram of the gel is shown withthe positions of molecular weight markersare indicated on the left (KaleidoscopePrestained Standard, Bio-Rad 161-0324)and right (Cross-Linked Phoshphorylaseb, Sigma P 8906). B, glycerol gradientanalysis of E1 oligomers induced by DNA.Radiolabled monomeric E1 (64 nM) wasincubated in the presence or absence of asingle-stranded DNA 10-base oligomer(10-mer, 500 nM) at room temperature for20 min in 25 mM KPO4 (pH 7.5), 75 mM

NaCl, 5 mM EDTA, 1 mM DTT, and 1mg/ml bovine serum albumin. Glutaral-dehyde was added to a 0.02% final con-centration and incubated for 20 min atroom temperature. Glutaraldehyde wasremoved from the reactions by SephadexG-25 column chromatography (NAP-10column, Pharmacia 17-0854-02). The pro-tein containing fractions were pooled foreach reaction and subjected to centrifuga-tion through gradients of 15–37% glycerolas described under “Materials and Meth-ods.” Fractions were collected an theamount of radioactivity in each fractionwas determined by scintillation. A graphof the radioactivity plotted against posi-tion in the gradient is shown. The posi-tions of molecular weight markers run ina parallel gradient is indicated at the topof the plot. C, the low and high molecularweight peaks from the glycerol gradientsin B were boiled in SDS sample buffer 1b-mercaptoethanol and analyzed by de-naturing gradient gel electrophoresis(4–10% acrylamide) and stained withCoomassie Brilliant Blue to allow identi-fication of molecular weight markers.This was followed by drying of the gel andautoradiography. Two autoradiogramsare presented representing two lengths ofexposure time. H (lanes 2 and 4) repre-sents the high molecular weight E1 spe-cies isolated from the glycerol gradient inB, and L (lanes 3 and 5) represents thelow molecular weight E1 peak. The lanemarked M (lane 1) is the Coomassie-stained cross-linked phosphorylase b mo-lecular weight marker (Sigma P 8906)with the weights indicated to the left. Tothe right, the positions of additional mo-lecular weight markers (KaleidoscopePrestained Standard, Bio-Rad 161-0324)are indicated.

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be continuously variable. The images contained in the 6- and3-fold averages were then sorted based upon the strength ofthis central density. Fig. 8a shows an average of 400 6-foldsymmetric rings with a strong hole near the center, while Fig.8b shows an average of 278 6-fold symmetric rings with a“plug” of density in the center. Similarly, Fig. 8c shows anaverage of 56 3-fold symmetric rings with a strong hole nearthe center, and Fig. 8d shows an average of 44 3-fold symmetricrings with a plug in the center. There did not appear to be anycorrelation between the strength of the 3- or 6-fold power and

the strength of the central density. Furthermore, both therelative strength of the 3-fold rotational power and the relativestrength of the central density appeared to be continuouslyvariable parameters. Thus, the groupings that are shown inFig. 8 represent arbitrary divisions. For example, averagescould have been created in Fig. 8, b and d, showing a strongercentral density by using fewer images, just as averages couldhave been generated in Fig. 8, c and d, showing a slightlystronger 3-fold power by using fewer images.

Symmetrized versions of the averages in Fig. 8, a-d, areshown in Fig. 8, e-h, respectively. One consequence of thesymmetrization, which eliminates noise, is that asymmetricfeatures disappear. However, this can also obscure real asym-metric features. The central holes in Fig. 8, a and c, are dis-placed from the central axis, while the symmetrization forcesthese holes to lie on the central axis in Fig. 8, e and g. It is likelythat this displacement of the stain-filled hole from the centralaxis is due to the binding of the 60-nucleotide oligomer, sincethis mass might be expected to be bound within the centralchannel to only one or two of the subunits, as shown for the T7gp4 hexameric helicase (10). Thus, the bound oligonucleotidewould be filling some of the central channel (indicated byarrows in Fig. 8, a and c), leading to an asymmetric location ofthe stain-filled hole.

While the averages in Fig. 8 have a slight hand, the degree ofchirality is much less than that observed for DnaB (39, 45), T7gp4 (10), or SV40 large T antigen (37). We therefore checked tosee if the lack of a strong hand arose from averaging togetherindividual projection images that were related by mirror sym-metry. This would occur if the rings were randomly oriented onthe grid, as opposed to predominantly one side adsorbing to thegrid. We used the highly chiral average of SV40 large T (37) asa reference to align all images, and then did the alignmentagainst the mirror image of the reference. Images were rankedbased upon the coefficient of correlation against the reference,and averages were then created from those with the strongestcorrelation using the reference-free alignment (44). The resultsof this procedure suggested that there was no strong chiralitypresent, as no averages were generated with the opposite bandto that shown in Fig. 8. Thus, the rings appear to predomi-nantly adsorb to the grid by the same surface.

Conditions were found where “side” views of the E1 ringscould be obtained by binding them to double-stranded DNAmolecules in the presence of ATP (Fig. 7b). Fig. 7c shows anaverage generated by aligning 217 side view images, eachimage containing three rings. The spacing between adjacentrings is about 50 Å, and unless there is a large degree ofinterdigitation, this would be the thickness of each ring. Sincethere does not appear to be a large continuous density runningbetween adjacent rings, a large degree of interdigitation ap-pears unlikely. The spacings between the rings were observedto be quite variable, and a number of different characteristicside views appeared to be present, as well. We therefore sortedthe images into subgroups using correspondence analysis (47)and Fig. 7, d and e, show subaverages generated from 44 and 52different rings, respectively. Since the alignment method usedto generate the averages in Fig. 7, d and e, only examined thedensity of the central ring (of the three contained in eachimage) the density of the central ring is averaged properly,while the density of the surrounding rings is smeared due totheir variable spacing. Nevertheless, the spacing of about 80 Åseen in Fig. 7d between the two outer rings suggests that theserings may be as close together as 40 Å. These side views helpestablish that the E1 ring is about 40–50 Å in thickness.

FIG. 5. Oligomerization of E1 correlates with helicase activity.A, titration of radiolabled E1 monomers (3.75, 7.5, 15, 30, 60, 120, 240,and 480 nM) into reactions containing 25 mM KPO4 (pH 7.5), 75 mM

NaCl, 5 mM EDTA, 1 mM DTT, 1 mg/ml bovine serum albumin, and a429-base pair BPV-1 origin containing DNA fragment (4.2 nM) for 20min at room temperature. Glutaraldehyde was added to a 0.04% finalconcentration and incubated for 20 min at room temperature. Reactionswere boiled in SDS sample buffer 1 b-mercaptoethanol and equalamounts of E1 protein loaded onto a denaturing acrylamide gradient gel(4–10%). Autoradiography was performed on the dried gel. The posi-tions of prestained molecular weight markers are indicated. Cross-linked radiolabled E1 hexamer (408 kDa) purified from a glycerol gra-dient (see Fig. 6) was loaded as a mass marker (lane 1). B, fragmentunwinding assay. The helicase assay was performed with the same E1titration and DNA levels as above. The DNA fragment is the same as inA and is radiolabled, while the E1 protein underwent a mock radiola-beling reaction with cold ATP. The resulting DNA products were as-sayed by agarose gel electrophoresis and autoradiography. The lanelabeled boiled provides markers for the ssDNA and double-strandedDNA positions. C, the amount of denatured DNA was determined byPhosphorImager analysis and is plotted against E1 protein concentration.

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DISCUSSION

Although the site-specific DNA-binding domains of the pap-illomavirus E1 have no homology to the SV-40/polyoma large Tantigens, both of which are highly homologous to each other,and the nucleotide sequence motifs that serve as binding sitesare distinct for these papillomaviruses; the initiator proteinsassemble as helicases in remarkably similar ways. In bothsituations a double-ringed structure assembles at the originsite, and this helicase activity is capable of denaturing DNAbidirectionally from the assembly point. As we have shownhere the toroidal rings formed by BPV-1 E1 are hexameric asare the T antigens.

It is also significant to point out that some differences havebeen uncovered between these helicases, particularly in theassembly pathways. Monomers of the T antigens can formhexameric complexes solely upon incubation with ATP andMg21; in contrast, as we show here E1 must bind DNA in orderto assemble as a hexamer. Similar observations have recentlybeen reported by Sedman and Stenlund (48), who have shownthat single strand DNA can initiate hexamer formation. Thatduplex DNA can also affect this multimerization fits nicely intoa pathway through which the enhancer protein E2 helps targetE1 to the ori site and once E2 frees itself from the preinitiationcomplex (27) double hexamers may readily follow at appropri-ate E1 concentrations.

These apparent biochemical differences may, however, bediscussed in another way that brings the papillomaviruses’mode of DNA replication even closer to the SV40/polyoma fam-ily. For DNA replication in vivo both SV40 and polyoma largeT antigens have enhancer sequences as cis-dominant elementsand for polyoma virus these elements are absolutely required.Interestingly, the polyomavirus large T antigen can bind coop-eratively with c-Jun, a factor naturally found to bind to poly-oma DNA, and this targeting stimulates helicase activity (49).Furthermore, E2 can activate polyoma virus replication in thecell if E2 sites are engineered into viral vectors (50). Althoughit is perhaps too premature to speculate on the evolutionarypathway through which the genes encoding for these viralinitiators descend, at least in part because we do not know howeukaryote chromosomal replication origins engage or assembleactive helicases, it seems possible that the special relationshipthat E2 and E1 have with each other mimics cellular processescaptured by the SV40/polyomavirus family.

Image Analysis of the Rings Reveals an Unexpected Hetero-geneity—Electron microscopy and image analysis have shownthat the rings formed by the E1 protein are hexameric. How-ever, the population of such rings formed in the presence of anoligonucleotide are not homogeneous, and two parameters of

Fig. 6. E1 complex bound to origin DNA. A, electron micrographs ofpurified E1 bound to a 242-base pair DNA fragment containing acentrally located BPV-1 origin sequence. Samples were prepared andspread as described under “Materials and Methods.” Bar, 100 nM. B,measurements of DNA lengths with or without bound E1 protein wereperformed by projecting electron micrograph negatives onto a Numon-ics digitizing tablet. The length distributions of 17 DNA moleculeswithout E1 and 55 molecules with bound E1 are shown. The distribu-tions are identical indicating that binding of E1 does not linearlycompact the DNA. C, models for E1 DNA binding. Binding modes arepresented in which the length of DNA would be unchanged upon bind-ing of a hexameric protein ring. Examples are also provided where theDNA is wrapped around the helicase ring resulting in linear compactionof the nucleic acid. The predicted changes in DNA length are shown.These calculations are based on the dimensions of the E1 hexamericring provided by three-dimensional image reconstruction (145 Å maxi-mum diameter 3 50 Å thickness) and the length of the 242-base pairB-DNA being 823 Å. (standard deviation for DNA length 5 9% 5 74 Å5 22 base pairs).

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variability were observed. First, a subset of rings existed not assymmetric hexamers, but as trimers of dimers, generating a3-fold rotational symmetry. This has previously been observedby electron microscopy for the hexameric rings formed by thednaB protein (39, 45) and the RecA protein (51). It is likely thatthis structural dimerization correlates with the biochemicalnon-equivalence of subunits observed for other hexameric he-licases. The T7 gp4 hexamer, for example, has been shown tocontain only three, not six, high affinity ATP-binding sites (52),as has the hexameric rho protein (53) and DnaB (54).

Second, a large mass of density can exist in the centralchannel of the ring, and appears to not depend upon whetherthe ring has 6- or 3-fold symmetry. What gives rise to thisdensity? We think it very unlikely that this density could arisefrom the oligonucleotide used to induce ring formation. Oneprimary reason is that this mass appears too large to be due tothe approximately 20-kDa mass of the oligonucleotide. Themass also appears to be continuously variable in its strength,an observation not compatible with it arising from the boundoligonucleotide. Also, since this oligonucleotide is required forring formation, it is hard to explain the existence of ringswithout this density if the density is due to the oligonucleotide.Third, this mass appears to be found on the rotational axis ofthe rings (Fig. 8, b and d), while we would expect the densitydue to the oligonucleotide not only to be much smaller butasymmetrically displaced from the central axis (10). Basedupon our experience imaging the T7 gp4 helicase with a boundoligonucleotide (10), the weak asymmetric density that is foundwithin the central channel in the E1 rings shown in Fig. 8, aand c, is consistent with the density that we would expect fromthe oligonucleotide.

Since the preparation is at least 95% pure (as judged bySDS-PAGE), and plugged centers were observed for E1 purifiedfrom both E. coli and SF9 cells, and the density is too great tobe due to the oligonucleotide, the most likely explanation isthat this central density arises from a portion of the E1 protein.This density may therefore arise from a disordered or highlymobile domain of the E1 protein, that can exist in multipleconformations. The recent crystal structure of an E. coli heli-case, the Rep protein (55), provides a possible clue in thisregard. Two copies of Rep were observed in the crystal, and thetwo differed by a rotation of the 2B subdomain by 130°. Sinceall helicases, including papilloma E1, are highly likely to have

a conserved structure (55, 56), the highly mobile subdomain inRep provides support for the possibility that the variable cen-tral density in E1 may be due to the large movement of such adomain.

Observations of the related SV40 large T antigen helicase3

have also shown a similar, variable central density. It is note-worthy that a three-dimensional reconstruction of large T an-tigen with a hole in the center (37) only accounted for about60% of the expected molecular volume, perhaps due to the factthat a portion of the subunit is mobile or disordered and notseen in the averaged reconstruction. A recent study also usedelectron microscopy to address the multimeric state of E1. Liuet al. (57) estimated from molecular volumes that E1 complexeson DNA are either hexameric or dihexameric. We did not findsuch size heterogeneity on DNA templates and it is possiblethat the hexamers observed by Liu et al. were actually inter-mediates or aggregates not detected in our experiments.

The Path of DNA through the Double Rings—The electronmicroscopic images of E1 assembled on duplex DNA (Fig. 6) areconsistent with the idea that the DNA somehow passes throughthe central cavities of the rings. Our end to end length meas-urements of the DNA fragments so engaged with the helicasedo not indicate a shortening and as shown in Fig. 9 this data byitself would be consistent with two sorts of models for strandpassage. In one model both strands might pass through thecenters of the double hexamers as shown in Fig. 9A. In thepresence of a single strand binding (SSB) protein and ATPunwinding might proceed either with the helicase working as amolecular motor translocating along the DNA and unwindingin opposite directions, or as a molecular pump denaturing theduplex and forcing it out through a central port. This class ofmodels is the one that seems to fit most of the data gathered forthe SV40 large T antigen. The DNase I and chemical protectiondata (obtained prior to SSB addition) argues in any case thatboth strands are protected. Moreover Dean et al. (58) haveshown that preformed hexamers of large T antigen are inactivefor unwinding of circular duplex DNA. We have made similarobservations for the BPV-1 E1 protein (data not shown). Theseresults would be consistent with models that required a topo-

3 X. Yu and E. H. Egelman, unpublished data.

FIG. 7. Electron micrographs of theE1 protein rings after incubationwith an oligonucleotide (a) or withdouble-stranded DNA and ATP (b).The 500-Å scale bar in a applies to both aand b. Averaged side views of the rings inb are shown in c-e. The average in c con-tains 217 images, while the subaveragesin d and e contain 44 and 52, respectively.The 100-Å scale bar in d applies to c-e.The double arrow in d is 80 Å long, indi-cating the approximate spacing betweenthree adjacent rings.

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logical link between the helicase ring and the circular DNA,and that stable hexamers once formed could not engage thecircle.

Curiously, preformed hexamers can displace single-strandedoligonucleotides annealed to circular single-stranded molecules(58).4 This result perhaps suggests that a hexameric ring mightengage the single strand circle from an external binding site,track along the DNA and upon engaging a duplex region passthe other strand through the center of the ring upon cycles ofhelicase action. Faced with duplex DNA the hexamer may haveno such entry and therefore a complex assembly process start-ing from monomers would create such possibilities for engage-ment. Such a model might predict a strand passage situationfor duplex DNA as depicted in Fig. 9B. The electron microscopicdata presented here would also be consistent with this notion.Considerable variation in lengths for melted or single-strandedDNA have been found, and the channels created between sub-units of the hexamers could space a strand as close to itscomplement in this arrangement as in the situation whereinboth strands passed through the center of the rings. (Compare

the positions of the black dots in the cross-sections shown inFig. 9, A and B.) In the model shown in Fig. 9B both singlestrands might be protected from DNase protection by positingthat the external one is buried or wrapped in the channel.Gillette et al. (59) have concluded from their results of DNAprotection experiments that E1 binding does produce one typeof complex resulting in DNA distortions even in the absence ofATP. Thus protein binding and assembly of the hexameraround DNA may provide enough energy to allow for one cycleof denaturation. It is also possible that the initial complexesdepicted in Fig. 9, A and B, are in some equilibrium with eachother and SSB or ATP might be expected to change thisdistribution.

Studies with the hexameric replication helicases from E. coliand their T phages have shown that a single subunit contactsthe single strand (at a given time) and that the other strandpasses outside of the ring (10, 60). In models for helicase action,this internal strand might be passed from one subunit to thenext in cycles of ATP hydrolysis. Thus an attraction of themodel in Fig. 9B is that it would lead to a conserved mode ofaction for the animal viral DNA helicases and their prokaryoterelatives.4 E. Fouts and M. R. Botchan, unpublished observations.

FIG. 8. The E1 rings appear with either a hole in the center (a and c) or with a plug of density in the center (b and d). Independentof whether there is a hole or a plug in the center, the rings have either a 6-fold symmetry (a and b) or a 3-fold symmetry (c and d). The images ine and f are the averages in a and b, respectively, but with an exact 6-fold symmetry imposed, while the images in g and h are the averages in cand d, respectively, but with an exact 3-fold symmetry imposed. The number of individual ring images in the averages shown is: a, 400; b, 278;c, 56; and d, 44. The arrows in a and c indicate a weak density that is located asymmetrically in the central channel, consistent with what mightbe expected from the bound oligonucleotide. The scale bar in h is 100 Å. The rotational power spectrum (63) for the average of 774 E1 rings showinga 6-fold rotational symmetry (with no 3-fold) (i), and the rotational power spectrum for an average of 100 E1 rings showing a significant 3-foldrotational symmetry (j). The spectra were calculated between the radial limits of 12 to 68 Å.

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Acknowledgments—We thank Arne Stenlund for providing the E. coliexpression system for E1, Seth Harris and James Berger for a criticalreading of the manuscript, and Terri DeLuca for word processing.

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E1 Hexameric Helicase4458

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Erik T. Fouts, Xiong Yu, Edward H. Egelman and Michael R. BotchanBiochemical and Electron Microscopic Image Analysis of the Hexameric E1 Helicase

doi: 10.1074/jbc.274.7.44471999, 274:4447-4458.J. Biol. Chem. 

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