Simian Virus 40 DNA Replication - Europe PMC

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Present address: Lederle Laboratories, American Cyanamid. Co., Pearl River, NY 10965. 73, 77); furthermore, in the case of the region involved in specific DNA ...
JOURNAL OF VIROLOGY, OCt. 1989, p. 4181-4188 0022-538X/89/104181-08$02.00/0

Vol. 63, No. 10

Copyright © 1989, American Society for Microbiology

Large T-Antigen Mutants Define Multiple Steps in the Initiation of Simian Virus 40 DNA Replication IAN J. MOHR,t* MICAELA P. FAIRMAN,4 BRUCE STILLMAN,

AND

YAKOV GLUZMAN§

Cold Spring Harbor Laboratory, Cold Spring Harbor, New York 11724 Received 9 March 1989/Accepted 12 June 1989

The biochemical activities of a series of transformation-competent, replication-defective large T-antigen point mutants were examined. The assays employed reflect partial reactions required for the in vitro replication of simian virus 40 (SV40) DNA. Mutants which failed to bind specifically to SV40 origin sequences bound efficiently to single-stranded DNA and exhibited nearly wild-type levels of helicase activity. A mutation at proline 522, however, markedly reduced ATPase, helicase, and origin-specific unwinding activities. This mutant bound specifically to the SV40 origin of replication, but under certain conditions it was defective in binding to both single-stranded DNA and the partial duplex helicase substrate. This suggests that additional determinants outside the amino-terminal-specific DNA-binding domain may be involved in nonspecific binding of T antigen to single-stranded DNA and demonstrates that origin-specific DNA binding can be separated from binding to single-stranded DNA. A mutant containing a lesion at residue 224 retained nearly wild-type levels of helicase activity and recognized SV40 origin sequences, yet it failed to function in an origin-specific unwinding assay. This provides evidence that origin recognition and helicase activities are not sufficient for unwinding to occur. The distribution of mutant phenotypes reflects the complex nature of the initiation reaction and the multiplicity of functions provided by large T antigen.

The replication of simian virus 40 (SV40) minichromosomes requires the presence of a functional large T antigen encoded by the virus and a permissive environment provided by the host cell (see references 8 and 76 for review). Genetic and biochemical studies have clearly demonstrated a role for this protein in the initiation of viral DNA replication (11, 43, 70, 71, 74); however, it has been postulated that T antigen also participates in the elongation of replication forks (69). Recently, large T antigen was shown to possess the ability to separate DNA duplexes into their component strands (68), which places it among a class of proteins known collectively as DNA helicases (25, 42). Monoclonal antibodies directed against large T antigen which inhibited unwinding in an in vitro replication system also inhibited the DNA helicase activity of purified T-antigen preparations (79). Large T antigen has also been reported to unwind plasmids containing a functional SV40 origin of replication in the presence of ATP, topoisomerase I, and other cellular factors (17, 82). This reaction requires the action of a DNA helicase and is similar to one known to take place at procaryotic origins of replication (2, 21). A notable difference is that origin recognition and DNA helicase functions, which are performed by several procaryotic proteins, are intrinsic properties of large T antigen, a single viral polypeptide. In addition, the association of large T antigen with DNA polymerase ao-primase activity in crude extracts (24, 65) is consistent with helicaseprimase interactions documented in procaryotic systems (see reference 47 for review). Mutational analysis of large T antigen has demonstrated that discrete regions of this multifunctional protein mediate different functions (13, 15, 38, 39, 45, 46, 52-55, 64, 66, 72,

73, 77); furthermore, in the case of the region involved in specific DNA binding, it has been possible to produce a truncated protein containing the N-terminal 259 amino acids which displays origin-specific DNA-binding activity (1, 72). The ability of large T antigen to direct the origin-specific replication of SV40 DNA, however, requires a virtually intact protein which possesses both specific DNA-binding and ATPase activities (13, 15, 45, 46, 56, 70). These functions have been delineated by both deletion analysis (13, 15, 55, 72, 77) and studies on transformation-competent, replication-defective point mutants (29-31, 45, 46, 53, 61, 73). Several of these mutants possess single-amino-acid substitutions which result in altered specific DNA-binding and/or ATPase activities. We used these mutants to examine the relationship, if any, between the DNA-binding, ATPase, helicase, and unwinding activities of large T antigen. MATERIALS AND METHODS Proteins. Mutant and wild-type T-antigen proteins were produced in HeLa cells coinfected with recombinant adenoviruses (producing either mutant or wild-type T antigen) and adenovirus type 5 as a helper (50, 63, 70, 71). Proteins were purified by immunoaffinity chromatography (63) with Pab419 (34) immobilized on protein A-Sepharose. Helicase assay. mpSV2 contains the HindIII-HpaII SV40 fragment (nucleotides 5171 to 346) cloned into Sma-HindIIIdigested M13mp9. A synthetic 42-basoligonucleotide (5'-

GTTTTCCCAGTCACGACGTTGTAAAACGACGGC CAGTGAATT-3') was synthesized on an Applied Biosystems 380-A DNA synthesizer and purified by electrophoresis on a denaturing polyacrylamide gel. Following adsorption

onto a reverse-phase C18 SepPak cartridge (Waters Associates), the oligonucleotide was eluted with 60% methanol, lyophilized to dryness, and suspended in H20. Then, 50 ng of purified oligonucleotide was annealed to 1.5 Lg of mpSV2 in 10 mM Tris hydrochloride (Tris-HCl, pH 8.5)-i mM MgCl2 at 50°C for 1 h. Following slow cooling to room temperature, the annealed template was labeled with the Klenow fragment of DNA polymerase I (in the presence of

Corresponding author. t Present address: Department of Molecular Biology, University of California, Berkeley, CA 94720. t Present address: Department of Zoology, University of Cambridge, Downing Street, Cambridge CB2 3EJ, England. § Present address: Lederle Laboratories, American Cyanamid Co., Pearl River, NY 10965. *

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[ot-32P]dCTP and nonradioactive dGTP) and subsequently extracted by both phenol and chloroform. Unincorporated nucleotides were removed by spinning the reaction mixture through a 1-ml Sepharose CL4B column, followed by ethanol precipitation of the DNA. Alternatively, the DNA was first ethanol precipitated, suspended, and loaded onto a CL4B column (0.6 by 26 cm). The column was equilibrated and developed in 10 mM Tris-HCl (pH 7.4)-i mM EDTA100 mM NaCl, and peak fractions containing labeled DNA were collected and used directly. Helicase reaction mixtures contained 10 ng of labeled substrate in 30 mM HEPES (N- 2- hydroxyethylpiperazine - N' -2- ethanesulfonic acid) KOH (pH 8.0)-4 mM ATP-7 mM MgCl2-0.S mM dithiothreitol and were incubated for 1 h at 37°C. Reaction were terminated by adding sodium dodecyl sulfate (SDS), EDTA, and glycerol to 0.6%, 30 mM, and 5%, respectively. Products were analyzed on 8% native polyacrylamide gels containing a TBE buffer gradient from 0.5 x to 2.5 x. Gels were dried onto DE81 paper (Whatmann) and exposed to Kodak XAR film at room temperature. DNA-binding reactions. Plasmid DNA templates containing site I (pOS-1; SV40 nucleotides 5171 to 5228), site II (pSVOdl3; SV40 nucleotides 5209 to 128), and the w.t. origin (pSVO+; SV40 nucleotides 5171 to 128) have been described before (70, 72). An equimolar mixture of these plasmids was assembled and digested to completion with TaqI. This enzyme releases intact SV40 origin sequences and generates several plasmid-derived fragments. The mixture was labeled with the Klenow fragment of DNA polymerase I, extracted with phenol and chloroform, and precipitated with ethanol; 50 ng of this mixture was incubated with various amounts of each T antigen in origin binding buffer (10 mM HEPES [pH 7.4], 100 mM KCl, 1 mM MgCl2, 5% glycerol, S0 jig of bovine serum albumin per ml) for 1 h at 0°C. Then, 5 jig of purified Pab419 (34) was added, and the incubation was continued for an additional 20 min. A 100-,ul amount of a 10% (vol/vol) protein A-Sepharose slurry in NET buffer (50 mM Tris-HCl [pH 7.4], 150 mM NaCl, 5 mM EDTA, 0.05% Nonidet P-40) containing sonicated calf thymus DNA at 20 jg/ml was then added, and the reaction mixes were incubated on a rocker for 50 min at 4°C. The beads were pelleted, washed three times with 1 ml of NET buffer, suspended in 1% SDS-25 mM EDTA, incubated at 65°C for 15 min, and electrophoresed on a 6% native polyacrylamide gel in a Tris-borate-EDTA buffer system. Gels were dried onto DE81 paper (Whatmann) and exposed to Kodak XAR film at room

temperature.

Partial duplex and single-stranded DNA-binding reactions

performed in helicase reaction buffer without ATP. Binding reaction mixes were incubated on ice for 1 h. Subsequent immunoprecipitation and wash steps were performed as described for origin-specific DNA-binding reactions, but unlabeled calf thymus DNA was not present in the protein A-Sepharose buffer. Pelleted protein A-Sepharose immune comp exes were counted directly (Cerenkov). To reduce background, it was helpful to first block nonspecific adsorption sites on the protein A beads. This was accomplished by incubating heat-denatured, sonicated calf thymus DNA (50 ,ug/ml) in the protein A-Sepharose-NET buffer slurry for 15 to 20 min at room temperature. The beads were then washed extensively with NET buffer to remove excess DNA prior to their addition to the reaction mixtures. Alternatively, samples were filtered through alkali-treated nitrocellulose (20, 48) and washed with 1 ml of NET buffer. Filters were dried and exposed to Kodak XAR film. Radioactive spots were excised from the filters and counted in liquid scintillant. were

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TABLE 1. Biochemical properties of transformation-competent,

replication-defective large T-antigen point mutantsa Mutant

C2 C6-2 C8A C1lA T22

Amino acid Ano(residue) acid change

Lys - Arg (516) Thr (153) Lys Glu (224) Pro Ser (522) His Gln (203)

Asn

Specific DNA-binding activityb

ATPase activity

_C -

+ +

+

+

+ -

+

APs

aSummarized from references 45, 46, and 70. b Binding of purified proteins to BstNI SV40 origin fragment. c C2 immunoprecipitated from extracts of transformed CV-1 cells bound to SV40 origin sequences (57). However, the specific DNA-binding activity of the C2 mutant in extracts from transformed CV-1 cells was reduced relative to that of equivalent amounts of wild-type T antigen present in extracts from COS-1 cells (Y.G. and M. Pizzolato, unpublished observations).

Origin-specific unvinding assay. Various amounts of purified SV40 large T antigen were added to reaction mixtures (50 jil) containing 40 mM HEPES-KOH (pH 7.5S)-8 mM MgCl2-0.5 mM dithiothreitol-4 mM ATP (from a 100 mM [pH 7.0] stock)-40 mM creatine phosphate-1 ,ug of creatine phosphokinase per ml-1 ,ug of bovine serum albumin-100 ng of topoisomerase I (purified from calf thymus)-1.2 jLg of RF-A (23, 81)-20 jLg of fraction SSI (23)-100 ng of template DNA (pSVO11). Reaction mixes were incubated at 37°C for 15 min, and reactions were terminated by the addition of SDS and EDTA to 0.4% and 100 mM, respectively. Protease XIII (50 jig/ml) and RNase A (10 jig/ml) were then added, and the samples were incubated for an additional 30 min at 37°C. The digested samples were then extracted once with an equal volume of phenol-chloroform (1:1), and the DNA was precipitated with ethanol. The degree of unwinding was determined by electrophoresis through 1.3% agarose gels in TBE and visualized both by staining with ethidium bromide and exposing the gel to X-ray film. RESULTS DNA helicase activity of mutant proteins. Table 1 presents a summary of the properties of several transformationcompetent, replication-defective point mutants. The helicase function of these mutants was evaluated by an assay similar to the one originally described by Venkatesan et al. (78) and used by Stahl et al. (68) in their studies on T antigen. The assay reflects the inherent ability of a DNA helicase to separate a partial duplex into its single-stranded components. The helicase substrate consists of an M13 phage (mpSV2) annealed to a synthetic 42-base oligonucleotide. This partial duplex was extended by the Klenow fragment of DNA polymerase I in the presence of [a-32P]dCTP and nonradioactive dGTP to produce a duplex region 51 nucleotides in length. Although this M13 clone contains some SV40 DNA sequences, only M13 DNA is present in the partial duplex region, while the SV40 origin region remains in single-stranded form. In the presence of ATP, Mg2+, and T antigen, the annealed 51-base oligonucleotide is displaced from the larger M13 DNA. The products of the reaction can then be resolved on native polyacrylamide gels and visualized by autoradiography. High-molecular-weight substrate DNA appears on the autoradiogram by virtue of its stable association with the labeled oligonucleotide. Progressive addition of large T antigen results in a decrease in the intensity of the substrate band with a concomitant increase in the intensity of the displaced oligonucleotide. The results of such an assay, done with saturating amounts of the

VOL. 63, 1989

PROPERTIES OF SV40 LARGE T-ANTIGEN MUTANTS

.

.

.

"

I .,

T

-2

!

K

_.t

-

_-_

FIG. 1. Helicase activity of mutant T antigens. A 1-,ug amount of each T antigen was incubated with the helicase substrate in the presence of ATP for 1 h at 37°C. Following electrophoresis on 8% native polyacrylamide-TBE buffer gradient gels, the reaction products were visualized by autoradiography. The lane marked 0 received no T antigen, and the lane marked BOIL received no T antigen and was boiled before being loaded on the gel. R115 is wild-type T antigen.

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--- -

Pt, ..............................

:.

wild-type protein (see titration in Fig. 5), are shown in Fig. 1. Similar results have been obtained by others with the same set of mutants (C. Prives, personal communication). Wildtype T antigen, along with mutants C2, C6-2, and T22, clearly possessed DNA helicase activity. Quantitation of these data by counting the radioactivity in excised bands revealed that approximately 79 and 85% of the labeled fragment was displaced by R115 (wild type) and T22, respectively, while C2 and C6-2 functioned at slightly reduced efficiencies and displaced approximately 54 to 70% of the labeled material. The significance of the slightly elevated activity of T22 relative to the wild type is not clear; however, it was reproducibly observed. The fact that C2, C6-2, and T22 displayed helicase activity is intriguing in light of the fact that these proteins failed to bind specifically to fragments containing the wild-type SV40 origin of replication (Fig. 2) (45, 46, 70). An ATPase-defective mutant, CilA, failed to displace the labeled oligonucleotide above levels present in the control reaction mixes lacking T antigen. Although T antigen prepared from the mKSA cell line examined by Stahl et al. in their original description of this activity displayed reduced helicase and ATPase activity (68), the precise nature and location of the mKSA lesion have not been determined. CllA, on the other hand, carries a singleamino-acid substitution of Ser for Pro at residue 522, and this substitution dramatically reduces the ATPase activity of the protein (45, 46, 70). The helicase activity of C8A was observed to vary with the conditions of isolation. Elution of the C8A protein from immunoaffinity matrices with 40 mM triethylamine resulted in the displacement of 2% of the labeled fragment (data not shown), whereas the other proteins again displayed nearly wild-type levels of helicase activity under identical conditions. The 57% displacement demonstrated in Fig. 1 was obtained only with C8A prepared with 20 mM triethylamine. The Lys -- Glu substitution at amino acid 224 in C8A may contribute to the enhanced sensitivity of C8A helicase activity to this basic reagent. The origin-specific DNA-binding activity of C8A was not affected by 40 mM triethylamine (not shown). Interestingly, treatment of wild-type T antigen with the alkylating agent N-ethylmaleimide reportedly abolishes helicase activity without affecting specific DNA binding (17). The sensitivity of the helicase activity to these agents

FIG. 2. Origin-specific DNA-binding activity of mutant T antigens. Either 100 ng (lanes 1), 300 ng (lanes 2), or 1,000 ng (lanes 3) of each T antigen was incubated with an equimolar mixture of end-labeled DNA fragments (M). After 60 min at 0°C, T antigen was immunoprecipitated from the reaction, the bound DNA fragments were electrophoresed on a 6% native polyacrylamide gel, and the dried gel was autoradiographed. The line drawings depict the three SV40 origin DNA fragments used in the experiment.

suggests that it is particularly labile in comparison with the specific DNA-binding activity. DNA-binding properties of mutant proteins. The specific DNA-binding properties of these mutants were reexamined by using a mixture of DNA templates containing site I, site II, and the wild-type origin on separate DNA fragments. The DNA-binding data in conjunction with ATPase data (not shown) were used to confirm the integrity of our protein preparations. Furthermore, previous studies (45, 46, 57, 70) were performed on only wild-type fragments and did not address binding of large T antigen to the individual sites. The assay was performed by incubating the purified proteins with a mixture of end-labeled DNA composed of three origin fragments present in equimolar amounts (wild type, site I plus site II; I, site I only; II, site II only) as well as several nonspecific DNA fragments. Protein DNA complexes, once formed, were immunoprecipitated (49), and the DNA fragments bound were visualized on autoradiograms of polyacrylamide gels. Labeled DNA fragments coimmunoprecipitating with T antigen thus reflect the amount of stable protein DNA complexes present in the reaction mix. C2, C6-2, and T22 failed to bind specifically to SV40 DNA sequences, and T22 exhibited an increased amount of nonspecific DNA binding (Fig. 2). The increased binding to nonspecific DNA may be related to the slightly elevated levels of DNA helicase activity displayed by this protein. While C8A and C1lA specifically bound to the wild-type SV40 origin fragment, they exhibited a grossly altered binding pattern to origin fragments containing either only site I or only site II. Previous binding studies (70) demonstrated that C8A and CliA bound wild-type origin fragments with

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MOHR ET AL. 5000

R11520

11000

10000

4000

9000 8000

n 3000 :3 0

7000

E

O 6000 E 5000

u

2000

4000

1000

3000 2000

0

100

1000

300

1000

ng T antigen

FIG. 3. Helicase substrate-binding activity of mutant T antigens. Various amounts of each T antigen were incubated with the labeled helicase substrate (7,200 cpm) in the absence of ATP. After 60 min at 0WC, T antigen was immunoprecipitated, and the pelleted immune complexes were subjected to direct Cerenkov counting. Background was 200 cpm.

slightly reduced efficiency, and this was confirmed in the present study. It is now clear that the interaction of these proteins with the individual sites is affected to a much greater extent. Binding to the partial duplex helicase substrate by the mutant T antigens was examined by a variation of the above immunoprecipitation assay. Since only one labeled species was present, the amount of DNA bound was analyzed by direct Cerenkov counting of the radioactivity in the immune complexes. ATP was omitted from these reactions so as to prevent the displacement of the labeled single strand by the helicase activity of T antigen. Substrate binding increased in proportion to T antigen concentration for all proteins except C1lA (Fig. 3). In addition to demonstrating a pronounced defect for ATPase and helicase activities, this protein was also defective in binding to the partial duplex helicase substrate. C2, C6-2, C8A, and T22 all bound the substrate efficiently despite the fact that they all failed to bind specifically to the SV40 origin of replication. The same results were obtained when metabolically labeled, single-stranded, circular mpSV2 DNA was used in the binding assay (not

shown). Effect of isolation conditions on single-stranded DNA binding. Initially, proteins were eluted from the immunoaffinity matrix with 40 mM triethylamine. Once we were aware of the effect this condition had on the helicase activity of C8A, it was of interest to ascertain its affect on other mutants which lacked helicase activity. The investigate the effects of isolation conditions on the altered activity displayed by the C1lA protein, a preparation was eluted from the immunoaffinity matrix with 20 mM triethylamine (hereafter referred to as C11A20) and compared with the original protein isolate prepared with 40 mM triethylamine (hereafter referred to as C11A40). Similar preparations of T antigen (R11520 versus R11540) served to define the effect of these conditions on the wild-type protein. The small decrease in the single-stranded DNA-binding activity of R11540 relative to that of R11521 (Fig. 4) could be related to its reduced helicase activity (Fig. 5). Although saturating amounts of both R11540 and R11520 displaced 95 to 97% of the labeled fragment, the helicase activity of R11540 was reduced threefold relative to that of R11520 under nonsaturating conditions (Fig. 5; compare R11540 [lane 1] and R11520 [lane 1]). R11540, however, still

0

300

100

1000

ng T antigen

FIG. 4. Helicase substrate-binding activity of proteins exposed on high pH. Various amounts of C1lA or R115 T antigens prepared with either 20 mM (C11A20 and R11520) or 40 mM (C11A40 and R11540) triethylamine were incubated with the labeled helicase substrate in the absence of ATP. After 60 min at 0°C, T antigen was immunoprecipitated, and the pelleted immune complexes were subjected to direct Cerenkov counting.

possessed substantially higher levels of helicase activity than C11A20 or C11A40. While there was a marginal effect on binding to wild-type origin fragments, R11540 demonstrated an approximately threefold decrease in binding to origin DNA fragments containing either site I only or site II only (Fig. 6). Minor differences in helicase activity and specific DNA binding were also observed when C11A20 was com-

....

Q

.u

..

.mm ....

FIG. 5. Effect of high-pH treatment on the helicase activity of T antigen. Various amounts (lanes 1, 100 ng; lanes 2, 300 ng; lanes 3, 1,000 ng) of CllA or R115 T antigens prepared with either 20 mM (C11A20 and R11520) or 40 mM (C1lA40 and R11540) triethylamine were incubated with the labeled helicase substrate in the presence of ATP for 1 h at 37°C. The products of the reaction were resolved on native 8% polyacrylamide gels and visualized by autoradiography. Both substrate and product bands were excised from the gel and counted in liquid scintillant to determine the percent fragment displaced. Lane 0 received no T antigen; the lane marked BOIL received no T antigen and was boiled before being loaded on the gel.

VOL. 63, 1989

PROPERTIES OF SV40 LARGE T-ANTIGEN MUTANTS

4185

dip

m_

-

____ H -

_

~ _e

--~

----

r~~~~~~~~~~~~~~~~~~~L FIG. 6. Origin-specific DNA-binding activity of proteins exposed to high pH. Various amounts (lane 1, 100 ng; lane 2, 300 ng; lane 0, none) of C11A or R115 T antigens prepared with either 20 mM (CllA20 and R115 20) or 40 mM (CllA40 and R11540) triethylamine were incubated with a mixture of labeled DNA containing SV40 origin fragments in equimolar amounts (lane M). After 60 min at O0C, T antigen was immunoprecipitated, and the labeled DNA fragments bound were separated on native 6% polyacrylamide gels and visualized by autoradiography. The line drawings depict the three SV40 origin DNA fragments used in the experiment.

pared with C11A. While C11A levels of helicase activity, C11A20 was capable of producing minor levels of activity at least 10-fold down from that of wild-type R11520 (Fig. 5). A 100-ng amount of R11520 displaced 53% of the labeled fragment, whereas quantities in excess of this amount were saturating. Identical amounts of C11A20 and C11A40 failed to displace significant amounts of labeled fragment; moreover, amounts of C11A020 in excess of 1 ag would be required to achieve 50% displacement of the labeled fragment (Fig. 5). Although C11A(20 displayed higher levels of helicase activity than C11At , preparations of C11Ag20 and C11Aw40 both functioned at levels 5 to 10% of the wild-type level in ATPase assays (not shown) as measured under standard ATPase conditions (50, 70). High-pH treatment of the C1iA protein also caused a threefold reduction in specific DNA binding to individual site I and site II fragments (Fig. 6). However, the specific DNA-binding activity of C11A40 was not affected to the same extent as its single-stranded DNA-binding activity. Figure 4 demonstrates that C11A40 again failed to bind to the labeled helicase substrate. C11A20 , however, bound efficiently, deviating only slightly from the wild-type R115 20 curve. Similar results were obtained when nitrocellulose filter binding was used to assay helicase substrate binding. Origin-specific unwinding activity of mutant proteins. A different assay which requires the participation of a DNA helicase measures unwinding of a closed circular DNA template containing the SV40 origin in the presence of T antigen, RF-A (a eucaryotic single-stranded DNA-binding protein [23, 81]), and a topoisomerase. Unwinding of the template is reflected by the appearance of faster-migrating

FIG. 7. Origin-specific unwinding activity of mutant T antigens capable of binding to origin sequences. Either 0, 1, or 2 p.g of purified T antigen was incubated with the purified, labeled DNA products of an in vitro DNA synthesis reaction in the presence of RF-A, calf thymus topoisomerase I, fraction SSI, and ATP for 15 min at 37°C. The reaction products were analyzed on 1% agarose gels and visualized by autoradiography.

supercoiled forms of the labeled DNA in reactions programmed with wild-type T antigen (Fig. 7). The unwinding activity of mutant proteins capable of specifically recognizing the SV40 origin of replication is also shown in this figure. C11A20 failed to generate any underwound forms of the template, demonstrating a requirement for wild-type levels of helicase activity. C8A, however, also failed to generate any underwound forms of the template. This is surprising, as this mutant displayed nearly wild-type levels of helicase activity and was capable of binding to regions of the SV40 origin of replication known to support the unwinding reaction (16). While this and other studies have demonstrated that site II binding and helicase activity are necessary for origin-specific unwinding to occur, the lack of activity observed with C8A suggests that these conditions are not sufficient for unwinding to occur. DISCUSSION Large T antigen provides multiple functions required for the initiation of SV40 DNA replication. T antigen specifically recognizes DNA sequences contained within the SV40 origin of replication and assembles a multimeric nucleoprotein complex on the core origin in the presence of ATP (6, 18, 19). Limited unwinding of origin sequences accompanied by structural deformations in the A+T-rich region ensues (7). The ability of large T antigen to interact efficiently with single-stranded DNA may first come into play in this reaction. In the presence of RF-A, a eucaryotic single-stranded DNA-binding protein required for origin-specific unwinding and the initiation of DNA replication (23, 81), and a topoisomerase (which relieves the torsional strain generated when a closed circular DNA molecule is unwound), the helicase activity of T antigen is free to unwind the substrate extensively (17, 82). This partial reaction is presumably coupled to the movement of the replication fork (78). Several of these activities are sequestered in discrete domains on the T-antigen molecule, while others appear to be produced by specific interactions between them. The helicase activity of T antigen appears to be generated by interplay between the N-terminal DNA-binding domain (amino acids 132 to 246 [72; D. McVey and Y. Gluzman,

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submitted for publication]) and the more C-terminus-proximal region involved in ATPase function (9, 12-14). This is supported by several observations. CliA, an ATPase-defective mutant containing a lesion at residue 522, failed to exhibit helicase activity and demonstrated reduced levels of origin-specific DNA binding. C11A40 also showed a marked reduction in its ability to bind nonspecific single-stranded DNA and the partial duplex helicase substrate. The proline residue altered by the Cl1A mutation is conserved in large T antigens from other papovaviruses and may thus represent a critical structural determinant in the carboxy terminus of the protein. C6-2 and C8A both reproducibly displayed slightly reduced levels of helicase activity relative to wild-type T antigen, and the lesions responsible for their reduced activity lay within the origin-specific DNA-binding domain. Although the C8A mutation did not prevent site-specific DNA binding, it rendered the helicase activity sensitive to higher levels of triethylamine. This may reflect communication between amino- and carboxy-terminal domains via a sensitive hinge region connecting the segments. The results with the C8A mutation further suggest that in addition to proper origin recognition and helicase activity, other requirements exist for T antigen to unwind origincontaining plasmids. While the C8A mutation clearly affects the efficiency of specific DNA binding, it may also have an effect on a subset of the base-specific contacts made by the protein upon complexing with the SV40 origin. Alternatively, the mutation may affect protein-protein interactions between T-antigen subunits bound to DNA. It is clear that individual T-antigen monomers assemble a specific nucleoprotein structure at the SV40 origin, and this structure may function as a mobile DNA helicase. Inability to assemble such a structure or the inability of such a structure to convert from a static DNA-binding assembly to a dynamic mobile helicase could also create the C8A phenotype. The nature of these conformational changes and the regions of the protein involved in producing them remain to be elucidated. Other studies are also consistent with a role for N-terminal sequences in producing helicase activity, since some Nterminal deletion mutants exist which have reduced ATPase activity (13). The monoclonal antibody PablO8 (33) binds the extreme N-terminus of the molecule and partially inhibits helicase activity with no effect on ATPase activity (79). Furthermore, Pabl613 (3) binds to an epitope (amino acids 200 to 250) within the DNA-binding domain and inhibits ATPase and helicase activities by more than 65% without affecting specific DNA binding (79). The effects of high pH on the biochemical activity of certain mutants highlight the importance of isolation conditions on the properties of a given preparation. Independent denaturation of individual protein domains has been observed in other polypeptides (51). Furthermore, pronounced effects of single-amino-acid substitutions have been reported to affect both the native and denatured states (35, 36, 62). Mutating critical residues may result in proteins which are more or less easily denatured by treatments which have little effect on their wild-type counterparts. In the case of an extremely complex polypeptide such as T antigen, such

treatments

may

result in partial denaturation of particularly

labile regions, as evidenced by C11A40. High-pH treatment also produces similar, less extreme phenotypes in the wildtype protein (Fig. 4 to 7). Finally, these effects point out a potential source of discrepancy among different laboratories isolating T antigen under a variety of conditions, the relative effects of which have not been investigated. While the focus of many structure-function studies is to define a linear domain responsible for a particular activity,

J. VIROL.

emphasis is seldom placed on identifying key residues outside of the minimal domain which have far-reaching effects on its function. Folding of the intact protein could easily bring disparate regions of the linear polypeptide into proximity. The Cl1A mutation at residue 522 falls into this category. Far removed from the DNA-binding domain (residues 132 to 246), the CliA mutation affects the efficiency with which the protein binds to origin fragments containing site I or site II. Furthermore, C11A40 failed to bind singlestranded DNA. In addition, 12 mutant T antigens, containing lesions outside the DNA-binding domain which rendered them temperature sensitive for SV40 DNA synthesis, possess an altered specific DNA-binding activity (80). While one class displays more pronounced defects following incubation at the nonpermissive temperature, another class exhibits gross alterations under normal conditions. Many of these mutations map to the carboxy-terminal portion of the protein in the vicinity of the Cl1A mutation. tsA58 affects residue 438, while tsA1642 affects residue 453. The remainder of the mutations examined all mapped between amino acids 447 and 708, 361 and 447, or 261 and 361. This last class may include residues in the putative metal-binding domain (4), mutations in which are also known to diminish specific DNA-binding activity (1). Large T antigen binds both specifically and nonspecifically to double-stranded DNA (10, 58, 60, 75) and nonspecifically to single-stranded DNA (67). While the region of the protein which interacts specifically with the SV40 origin of replication is fairly well defined (39, 52, 64, 72), relatively little is known about the region involved in binding to singlestranded DNA. T antigen from SV80 cells was used in the original studies which examined single-stranded DNA binding (67). This T antigen was subsequently shown to contain a mutation at residue 147 which reduced its origin-specific DNA-binding activity (28). The Cl1A mutation raises the possibility that additional determinants for single-stranded DNA binding exist on the molecule outside of the specific DNA-binding domain. Whether they exist in a discrete segment of the protein or involve several key residues in isolated regions remains to be determined. In this regard, it is interesting that residues 372 to 648 of T antigen exhibit homology with the RecA protein (residues 36 to 352) from Escherichia coli (59). The RecA protein is an ATPase which binds to both single- and double-stranded DNA; in addition, it possesses a strand-separating activity (5, 37; for review, see reference 22). On the other hand, truncated T antigens containing amino acids 1 to 246 bind to the partial duplex helicase substrate, supporting the idea that a single DNAbinding domain exists on the molecule which recognizes both single- and double-stranded DNA (McVey et al., submitted). The putative metal-binding motif (residues 302 to 320) may also be involved in single-stranded DNA binding. Such an idea is supported by studies on bacteriophage T4 gene 32 protein, a single-stranded DNA-binding protein which requires zinc for its efficient interaction with DNA (26, 27, 40). Viral genomes bearing mutations at Cys or His residues in this region of T antigen fail to replicate and hence do not produce plaques (44). Finally, it is conceivable that critical residues outside the single N-terminal DNA-binding domain modulate the structure of this domain so as to favor single-stranded DNA binding over origin-specific DNA binding. This would not be without precedent, as the phosphorylation clusters which modulate both DNA replication and specific DNA binding lie outside the DNA-binding domain (32, 41, 50). Although further experiments are necessary to delineate precisely all of the regions of the protein involved in single-stranded DNA binding, it is clear that the ability of

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T antigen to bind to single-stranded DNA can be genetically and biochemically separated from its origin-specific DNAbinding activity. Analysis of these mutant T antigens lends support to the biological significance of these partial biochemical reactions and has allowed us to pinpoint their defects in the initiation of DNA replication. Furthermore, the distribution of their phenotypes reflects the complex nature of the initiation reaction and the multiplicity of functions performed by T antigen.

17. Dean, F. B., P. Bullock, Y. Murakami, C. R. Wobbe, L. Weissbach, and J. Hurwitz. 1987. Simian virus 40 (SV40) DNA replication: SV40 large T antigen unwinds DNA containing the SV40 origin of replication. Proc. Natl. Acad. Sci. USA 84: 16-20. 18. Dean, F. B., M. Dodson, H. Echols, and J. Hurwitz. 1987. ATP-dependent formation of a specialized nucleoprotein structure by simian virus 40 (SV40) large tumor antigen at the SV40 replication origin. Proc. Natl. Acad. Sci. USA 84:8981-8985. 19. Deb, S. P., and P. Tegtmeyer. 1987. ATP enhances the binding of simian virus 40 large T antigen to the origin of replication. J.

ACKNOWLEDGMENT This work was funded by Public Health Service grant CA 13106 from the National Cancer Institute to Cold Spring Harbor Laboratory.

Virol. 61:3649-3654. 20. Diffley, J. F. X., and B. Stillman. 1986. Purification of a cellular double-stranded DNA-binding protein required for initiation of adenovirus DNA replication by using a rapid filter-binding assay. Mol. Cell. Biol. 6:1363-1373. 21. Dodson, M., H. Echols, S. Wickner, C. Alfano, K. MensaWilmot, B. Gomes, J. LeBowitz, J. D. Roberts, and R. McMacken. 1986. Specialized nucleoprotein structures at the origin of replication of bacteriophage X: localized unwinding of duplex DNA by a six-protein reaction. Proc. Natl. Acad. Sci. USA 84:7638-7642. 22. Dressler, D., and H. Potter. 1982. Molecular mechanisms in genetic recombination. Annu. Rev. Biochem. 51:727-761. 23. Fairman, M. P., and B. Stillman. 1988. Cellular factors required for multiple stages of SV40 DNA replication in vitro. EMBO J. 7:1211-1218. 24. Gannon, J. V., and D. Lane. 1987. p53 and DNA polymerase a compete for binding to SV40 T antigen. Nature (London) 329:456-458. 25. Geider, K., and H. Hoffman-Berling. 1981. Proteins controlling the helical structure of DNA. Annu. Rev. Biochem. 50:233-260. 26. Geidroc, D. P., K. M. Keating, K. R. Williams, and J. E. Coleman. 1987. The function of zinc in gene 32 protein from T4. Biochemistry 26:5251-5259. 27. Geidroc, D. P., K. M. Keating, K. R. Williams, W. H. Konigsberg, and J. E. Coleman. 1986. Gene 32 protein, the singlestranded DNA-binding protein from bacteriophage T4, is a zinc metalloprotein. Proc. Natl. Acad. Sci. USA 83:8452-8456. 28. Gish, W. R., and M. R. Botchan. 1987. Simian virus 40transformed human cells that express large T antigens defective for viral DNA replication. J. Virol. 61:2864-2876. 29. Gluzman, Y. 1981. SV40-transformed simian cells support the replication of early SV40 mutants. Cell 23:175-182. 30. Gluzman, Y., and B. Ahrens. 1982. SV40 early mutants that are defective for viral DNA synthesis but competant for transformation of cultured rat and simian cells. Virology 123:78-92. 31. Gluzman, Y., J. Davison, M. Oren, and E. Winocour. 1977. Properties of permissive monkey cells transformed by UVirradiated simian virus 40. J. Virol. 22:256-266. 32. Grasser, F. A., K. Mann, and G. Walter. 1987. Removal of serine phosphates from simian virus 40 large T antigen increases its ability to stimulate DNA replication in vitro but has no effect on ATPase and DNA binding. J. Virol. 61:3373-3380. 33. Gurney, E. G., D. Tamowsky, and W. Deppert. 1986. Antigenic binding sites of monoclonal antibodies specific for simian virus 40 large T. J. Virol. 57:1168-1172. 34. Harlow, E., L. V. Crawford, D. C. Pim, and N. M. Williamson. 1981. Monoclonal antibodies specific for simian virus 40 tumor antigens. J. Virol. 39:861-869. 35. Hecht, M. R., J. M. Sturtevant, and R. T. Sauer. 1984. Effect of single amino acid replacements on the thermal stability of the NH2-terminal domain of phage K repressor. Proc. Natl. Acad. Sci. USA 81:5685-5689. 36. Hecht, M. R., J. M. Sturtevant, and R. T. Sauer. 1986. Stabilization of X repressor against thermal denaturation by sitedirected Gly -* Ala changes in a-helix 3. Proteins 1:43-46. 37. Iwabuchi, M., T. Shibata, T. Ohtani, M. Natori, and T. Ando. 1983. ATP-dependent unwinding of the double helix and extensive supercoiling by Escherichia coli RecA protein in the presence of topoisomerase. J. Biol. Chem. 258:12394-12404. 38. Kalderon, D., W. Richardson, A. Markham, and A. Smith. 1984. Sequence requirements for nuclear localization of simian virus 40 large T antigen. Nature (London) 311:33-38.

LITERATURE CITED 1. Arthur, A. K., A. Hoss, and E. Fanning. 1988. Expression of simian virus 40 T antigen in Escherichia coli: localization of T antigen origin DNA-binding domain to within 129 amino acids.

J. Virol. 62:1999-2006. 2. Baker, T. A., K. Sekimizu, B. E. Funnell, and A. Kornberg. 1986. Extensive unwinding of the plasmid template during

staged enzymatic initiation of DNA replication from the origin of the Escherichia coli chromosome. Cell 45:53-64. 3. Ball, R. K., B. Siegel, S. Quellhorst, G. Brandner, and D. G. Braun. 1984. Monoclonal antibodies against simian virus 40 nuclear large T tumor antigen: epitope mapping, papovavirus cross-reaction, and cell surface staining. EMBO J. 3:1485-1491. 4. Berg, J. M. 1986. Potential metal-binding domains in nucleic

acid-binding proteins. Science 232:485-487. 5. Bianchi, M., B. Riboli, and G. Magni. 1985. E. coli RecA protein

possesses a strand separating activity on short duplex DNAs. EMBO J. 4:3025-3030. 6. Borowiec, J. A., and J. Hurwitz. 1988. ATP stimulates the binding of simian virus 40 (SV40) large tumor antigen to the SV40 origin of replication. Proc. Natl. Acad. Sci. USA 85:

64-68. 7. Borowiec, J. A., and J. Hurwitz. 1988. Localized melting and structural changes in the SV40 origin of replication induced by T antigen. EMBO J. 7:3149-3158. 8. Botchan, M., T. Grodzicker, and P. Sharp (ed.). 1986. DNA tumor viruses: control of gene expression and replication (Cancer Cells, vol. 4). Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. 9. Bradley, M. K., T. F. Smith, R. H. Lathrop, D. M. Livingston, and T. A. Webster. 1987. Consensus topography in the ATPbinding site of simian virus 40 and polyomavirus large tumor antigens. Proc. Natl. Acad. Sci. USA 84:4026-4030. 10. Carroll, R. B., L. Hager, and R. Dulbecco. 1974. Simian virus 40 T antigen binds to DNA. Proc. Natl. Acad. Sci. USA 71: 3754-3757. 11. Chou, J. Y., and R. G. Martin. 1974. Complementation of simian virus 40 mutants. J. Virol. 13:1101-1109. 12. Clark, R., D. P. Lane, and R. Tjian. 1981. Use of monoclonal antibodies as probes of simian virus 40 T antigen ATPase

activity. J. Biol. Chem. 256:11854-11858. 13. Clark, R., K. Peden, J. M. Pipas, D. Nathans, and R. Tjian. 1983. Biochemical activities of T antigen proteins encoded by simian virus 40 A gene deletion mutants. Mol. Cell. Biol. 3:220-228. 14. Clertant, P., P. Gaudray, E. May, and F. Cuzin. 1984. The nucleotide binding site detected by affinity labeling in the large T proteins of polyoma and SV40 viruses is distinct from their ATPase catalytic site. J. Biol. Chem. 259:15196-15203. 15. Cole, C. N., J. Tornow, R. Clark, and R. Tjian. 1986. Properties of the simian virus 40 (SV40) large T antigens encoded by SV40 mutant with deletions in gene A. J. Virol. 57:539-546. 16. Dean, F. B., J. A. Borowiec, Y. Ishimi, S. Deb, P. Tegtmeyer, and J. Hurwitz. 1987. Simian virus 40 large tumor antigen requires three core replication origin domains for DNA unwinding and replication in vitro. Proc. Natl. Acad. Sci. USA 84: 8267-8271.

4188

MOHR ET AL.

39. Kalderon, D., and A. E. Smith. 1984. In vitro mutagenesis of a putative DNA binding domain of SV40 large T antigen. Virology 139:109-137. 40. Keating, K. M., L. R. Ghosaini, D. P. Geidroc, K. R. Williams, J. E. Coleman, and J. M. Sturtevant. 1988. Thermal denaturation of T4 gene 32 protein: effects of zinc removal and substitution. Biochemistry 27:5240-5245. 41. Klausing, K., K. H. Scheidtmann, E. A. Baumann, and R. Knippers. 1988. Effects of in vitro dephosphorylation on DNA binding and DNA helicase activities of simian virus 40 large tumor antigen. J. Virol. 62:1258-1265. 42. Kornberg, A. 1980. DNA replication. W. H. Freeman & Co., San Francisco. 43. Li, J. J., and T. J. Kelly. 1984. Simian virus 40 DNA replication in vitro. Proc. Natl. Acad. Sci. USA 81:6973-6977. 44. Loeber, G., R. Parsons, and P. Tegtmeyer. 1989. The zinc finger region of simian virus 40 large T antigen. J. Virol. 63:94-100. 45. Manos, M. M., and Y. Gluzman. 1984. Simian virus 40 large T antigen point mutants that are defective in viral DNA replication but competent in oncogenic transformation. Mol. Cell. Biol. 4:1125-1133. 46. Manos, M. M., and Y. Gluzman. 1985. Genetic and biochemical analysis of transformation-competent, replication-defective simian virus 40 large T antigen mutants. J. Virol. 53:120-127. 47. Marians, K. J. 1984. Enzymology of DNA replication in prokaryotes. Crit. Rev. Biochem. 17:153-215. 48. McEntee, K., G. M. Weinstock, and I. R. Lehman. 1980. RecA protein-catalyzed strand assimilation: stimulation by Escherichia coli single-stranded DNA-binding protein. Proc. Natl. Acad. Sci. USA 77:857-861. 49. McKay, R. D. G. 1981. Binding of a simian virus 40 large T antigen-related protein to DNA. J. Mol. Biol. 145:471-488. 50. Mohr, I. J., B. Stillman, and Y. Gluzman. 1987. Regulation of SV40 DNA replication by phosphorylation of T antigen. EMBO J. 6:153-160. 51. Pabo, C. O., R. T. Sauer, J. M. Sturtevant, and M. Ptashne. 1979. The X repressor contains two domains. Proc. Natl. Acad. Sci. USA 76:1608-1612. 52. Paucha, E., D. Kalderon, R. W. Harvey, and A. E. Smith. 1986. Simian virus 40 origin DNA-binding domain on large T antigen. J. Virol. 57:50-64. 53. Peden, K. W. C., and J. M. Pipas. 1985. Site-directed mutagenesis of the simian virus 40 large T antigen gene: replicationdefective amino acid substitution mutants that retain the ability to induce morphological transformation. J. Virol. 55:1-9. 54. Pintel, D., N. Bouck, and G. DiMayorca. 1981. Separation of lytic and transforming functions of the SV40 A region: two mutants which are temperature sensitive for lytic functions have opposite effects on transformation. J. Virol. 38:518-528. 55. Pipas, J. M., K. W. C. Peden, and D. Nathans. 1983. Mutational analysis of simian virus 40 T antigen: isolation and characterization of mutants with deletions in the T antigen gene. Mol. Cell. Biol. 3:203-213. 56. Polvino-Bodnar, M., and C. N. Cole. 1982. Construction and characterization of viable deletion mutants of simian virus 40 lacking sequences near the 3' end of the early region. J. Virol.

43:489-502. 57. Prives, C., L. Covey, A. Scheller, and Y. Gluzman. 1983. DNA-binding properties of simian virus 40 T antigen mutants defective in viral DNA replication. Mol. Cell. Biol. 3:1958-1966. 58. Reed, S. I., J. Ferguson, R. W. Davis, and G. Stark. 1975. T antigen binds to SV40 at the origin of DNA replication. Proc. Natl. Acad. Sci. USA 72:1605-1609. 59. Seif, R. 1982. New properties of simian virus 40 large T antigen. Mol. Cell. Biol. 2:1463-1471. 60. Shalloway, D., T. Kleinberger, and D. M. Livingston. 1980. Mapping of SV40 DNA replication origin region binding sites for the SV40 T antigen by protection against exonuclease III

digestion. Cell 20:411-422.

J. VIROL. 61. Shiroki, K., and H. Shimojo. 1971. Transformation of green monkey kidney cells by SV40 genome: the establishment of transformed cell lines and the replication of human adenoviruses and SV40 in transformed cells. Virology 45:163-171. 62. Shortle, D., and A. K. Meeker. 1986. Mutant forms of staphylococcal nuclease with altered patterns of guanidine hydrochloride and urea denaturation. Proteins 1:81-89. 63. Simanis, V., and D. P. Lane. 1985. An immunoaffinity purification for SV40 large T antigen. Virology 4:88-100. 64. Simmons, D. T. 1986. DNA-binding region of the simian virus 40 tumor antigen. J. Virol. 57:776-785. 65. Smale, S. T., and R. Tjian. 1986. T antigen-DNA polymerase a complex implicated in simian virus 40 DNA replication. Mol. Cell. Biol. 6:4077-4087. 66. Soprano, K. J., N. Galanti, G. J. Jonak, S. McKercher, J. M. Pipas, K. W. C. Peden, and R. Baserga. 1983. Mutational analysis of simian virus 40 T antigen: stimulation of cellular DNA synthesis and activation of rRNA genes by mutants with deletions in the T antigen gene. Mol. Cell. Biol. 3:214-219. 67. Spillman, T., D. Giacherio, and L. P. Hager. 1979. Single strand DNA binding of simian virus 40 tumor antigen. J. Biol. Chem. 254:3100-3104. 68. Stahl, H., P. Droge, and R. Knippers. 1986. DNA helicase activity of SV40 large tumor antigen. EMBO J. 5:1939-1944. 69. Stahl, H., P. Droge, H. W. Zentgraf, and R. Knippers. 1985. A large tumor antigen-specific monoclonal antibody inhibits DNA replication of simian virus 40 minichromosomes in an in vitro elongation system. J. Virol. 54:473-482. 70. Stillman, B., R. D. Gerard, R. A. Guggenheimer, and Y. Gluzman. 1985. T antigen and template requirements for SV40 DNA replication in vitro. EMBO J. 4:2933-2939. 71. Stillman, B. W., and Y. Gluzman. 1985. Replication and supercoiling of simian virus 40 DNA in cell extracts from human cells. Mol. Cell. Biol. 5:2051-2060. 72. Strauss, M., P. Argani, I. J. Mohr, and Y. Gluzman. 1987. Studies on the origin-specific DNA-binding domain of simian virus 40 large T antigen. J. Virol. 61:3326-3330. 73. Stringer, J. R. 1982. Mutant of simian virus 40 large T antigen that is defective for viral DNA synthesis, but competent for transformation of cultured rat cells. J. Virol. 42:854-864. 74. Tegtmeyer, P. 1972. Simian virus 40 deoxyribonucleic acid synthesis: the viral replicon. J. Virol. 10:591-598. 75. Tjian, R. 1978. The binding site on SV40 DNA for a T antigen-related protein. Cell 13:165-179. 76. Tooze, J. (ed.). 1981. Molecular biology of tumor viruses, part 2: DNA tumor viruses. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. 77. Tornow, J., and C. N. Cole. 1983. Nonviable mutants of simian virus 40 with deletions near the 3' end of gene A define a function for large T antigen required after the onset of viral DNA replication. J. Virol. 47:487-494. 78. Venkatesan, M., L. L. Silver, and N. G. Nossal. 1982. Bacteriophage T4 gene 41 protein, required for the synthesis of RNA primers, is also a DNA helicase. J. Biol. Chem. 257:1242612434. 79. Wiekowski, M., P. Droge, and H. Stahl. 1987. Monoclonal antibodies as probes for a function of large T antigen during the elongation process of simian virus 40 DNA replication. J. Virol. 61:411-418. 80. Wilson, V. G., M. J. Tevethia, B. A. Lewton, and P. Tegtmeyer. 1982. DNA-binding properties of simian virus 40 temperaturesensitive A proteins. J. Virol. 44:458-466. 81. Wold, M. S., and T. Kelly. 1988. Purification and characterization of replication protein A, a cellular protein required for in vitro replication of simian virus 40 DNA. Proc. Natl. Acad. Sci. USA 85:2523-2527. 82. Wold, M. S., J. J. Li, and T. J. Kelly. 1987. Initiation of simian virus 40 DNA replication in vitro: large tumor antigen and origin-dependent unwinding of the template. Proc. Nati. Acad. Sci. USA 84:3643-3647.