The murine p53 protein blocks replication of SV40 DNA in vitro by inhibiting the initiation functions of SV40 large T antigen

Cell, Vol. 57, 379-392,

May 5, 1989, Copyright

0 1989 by Cell Press

The Murine ~53 Protein Blocks Replication of SV40 DNA In Vitro by Inhibiting the Initiation Functions of SV40 Large T Antigen Edith H. Wang, Paula N. Friedman, Department of Biological Sciences Columbia University New York, New York 10027

and Carol Prives

Summary We have characterized the effect of murine p53 on SV40 DNA replication in vitro. Purified wild-type murine p53 dramatically inhibited the ability of SV40 Tantigen to mediate the replication of a plasmid bearing the viral origin (o&DNA) in vitro. In contrast, polyoma o&DNA replication in vitro was unaffected by ~53. Surprisingly, both unbound p53 and SV40 T antigen-bound p53 were equally detrimental to SV40 oriDNA replication. Thus, ~53 interferes with interactions between T antigen molecules that are required for DNA synthesis. p53 inhibited the binding to and subsequent unwinding of the SV40 origin by T antigen and thus selectively blocked the initial stages of oriDNA replication. In contrast to the nononcogenic wild-type murine ~53, high concentrations of a mutant transforming p53 failed to block SV40 ori-DNA replication in vitro. These observations may provide insight into a possible role for p53 in the cell. Introduction SV40 large T antigen functions are required for oncogenic cell transformation and viral DNA replication (for review, see Tooze, 1981; Fried and Prives, 1986). In most cells that express the SV40 early region, a fraction of the virally encoded large T antigen is bound to the cellular protein, ~53 (for review, see Oren, 1985). ~53 itself has been shown by a variety of approaches to affect the proliferation of primary cells (Eliyahu et al., 1984; Jenkins et al., 1984; Parada et al., 1984) although it appears that mutant but not wild-type ~53 proteins have oncogenic function (Hinds et al., 1989). It is therefore possible that one of the modes by which SV40 T antigen alters the growth properties of cells is through its interaction with ~53. Conversely, ~53 might affect one or more of the replicative activities of T antigen, perhaps reflecting its function in the uninfected cell. Earlier studies on SV40 DNA synthesis provided evidence that large T antigen is required for the initiation of viral DNA replication (Tegtmeyer, 1972; Chou et al., 1974), while more recent experiments suggest that the viral product is also required for DNA chain elongation (Stahl et al., 1985; Wiekowski et al., 1987; Wobbe et al., 1987). It has been established that T antigen binds specifically and with high affinity to the viral replication origin (Tegtmeyer et al., 1983; Jones and Tjian, 1984). Although the precise dissection of the early stages of viral DNA synthesis has not yet been accomplished, it has been shown that T antigen can specifically unwind DNA containing the viral repli-

cation origin (Dean et al., 1987a; Wold et al., 1987). Related to this are the facts that T antigen exhibits both ATPase (Giacherio and Hager, 1979; Tjian and Robbins, 1979) and helicase (Stahl et al., 1986) activities. These two properties, coupled with the ability of T antigen to bind to DNA polymerase a (Smale and Tjian, 1986) may explain its functions in the initiation and elongation of viral DNA replication. In contrast to T antigen, a specific biochemical activity has not yet been identified for ~53. It has been established, however, that ~53 is a phosphoprotein located in the nucleus of some but not other cells (Rotter et al., 1983; Milner and Cook, 1986; Zajdel and Blair, 1988). While relatively stable in many types of transformed cells, its half-life in normal cells is short, on the order of minutes (Oren et al., 1981). In addition to SV40 T antigen, p53 binds to the 58 kd protein encoded by the adenovirus ElB region (Sarnow et al., 1982). Moreover, some forms of ~53 bind to the heat shock cognate protein hsc70 (Clarke et al., 1988; Finlay et al., 1988) and when expressed in E. coli bind to the dnaK gene product, also a heat shock protein (Clarke et al., 1988). Because transformed cell lines that do not express ~53 have been identified (Benchimol et al., 1982; Mowat et al., 1985; Wolf et al., 1985) its function is not an absolute requirement for the viability of all cells. However, normal cells fail to progress from G O to S phase when injected with anti-p53 antibodies (Mercer et al., 1982), and when induced to express ~53 antisense RNA proliferate more slowly than their control cell counterparts (Shohat et al., 1987). Taken together, these studies suggest that ~53 is a protein involved in regulating cell growth, possibly playing a role in the control of cellular DNA replication. The development of systems for the synthesis of DNA bearing the SV40 (Li and Kelly, 1984; Stillman et al., 1985; Wobbe et al., 1985) or the polyoma (Murakami et al., 1986a) origin in vitro has provided the opportunity to study both the functions of the large T antigens encoded by these viruses and the cellular factors involved in DNA replication. To understand how ~53 might affect either viral or cellular DNA synthesis, we have analyzed the effects of the host protein on SV40 and polyoma T antigen-dependent DNA replication in vitro. Results SV40 T Antigen but Not Polyoma T Antigen Forms a Stable Complex with Murine p53 In Vivo To study the interactions of ~53 and T antigen, we utilized baculoviral vectors vEV55SVT and vEV55p53, which encode wild-type SV40 T antigen and wild-type murine ~53, respectively @ ‘Reilly and Miller, 1988), as well as vEV51LT, which encodes wild-type polyoma large T antigen (Rice et al., 1987). SV40 large T antigen and ~53 proteins synthesized in Sf27 insect cells form a stable complex, as demonstrated by coimmunoprecipitation with their respective monoclonal antibodies and cosedimentation on sucrose gradients (C)‘Reilly and Miller, 1988). To confirm

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Frgure 1 Expressron

of SV40 and Polyoma T Antigens

and Murtne p53 rn Insect Ceils

(A) s5S-labeled extracts prepared from Sf27 cells infected with recombrnant baculovrrus vectors expressmg SV40 T antrgen (SV), polyoma T antrgen (Py), or murine p53 (p53), or coinfected with vectors expressrng SV40 T antigen and murrne p53 (SV + ~53) or polyoma T antigen and rmurrne ~53 (Py + ~53) were immunoprecipitated with SV40 T antigen monoclonal PAb 419 (lanes 1. 4, 5) p53 monoclonal PAb 421 (lanes 2, 3, 6, 9, ll), or polyoma T antigen monoclonal PAb F4 (lanes 7, 8, 10). The immunoprecipitates were analyzed by SDS-PAGE and autoradiography (B) Sf27 cells were Infected with recombinant baculovrruses encoding either SV40 T antrgen. polyoma T antigen, or murine p53 followed by cmmunoaffrmty purifrcatron with the appropriate monoclonal antrbody and analysis of the protems by SDS-PAGE and stlver starmng Quantities of SV40 T anttgen (lane l), polyoma T antigen (lane 2) and p53 (lane 3) were eshmated as 15. 04. and 1.0 trg, respectively, by comparrson with standards.

and extend this observation, Sf27 insect cells were infected with recombinant baculoviruses expressing SV40 T antigen and murine p53 individually or together. Lysates prepared from [35S]methionine-labeled cells infected with SV40 and/or p53 vectors were immunoprecipitated with either anti-SW0 T antigen monoclonal antibody PAb 419 or anti-p53 monoclonal antibody PAb 421 (Harlow et al., 1981; Figure 1A). SV40 T antigen was efficiently immunoprecipitated by PAb 419 but not PAb 421, and p53 with PAb 421 but not PAb 419. However, in lysates derived from cells coinfected with the two recombinant baculoviruses, SV40 T antigen and ~53 were coprecipitated with either PAb 419 or PAb 421. Since neither antibody cross-reacts with the reciprocal protein (Harlow et al., 1981) the ability of both proteins to be immunoprecipitated with either antibody must

result

from

the

formation

of a stable

complex

be-

tween SV40 T antigen and ~53. Although SV40 and polyoma T antigens share extensive homology (Soeda et al., 1980; Tooze, 1981) cells expressing the polyoma early region do not normally contain high levels of ~53. To determine whether or not polyoma T antigen and ~53 form a complex, Sf27 cells were infected with recombinant baculoviruses expressing polyoma large T antigen and murine ~53. Lysates from cells coinfected with the p53 and polyoma vectors were immunoprecipitated with either PAb 421 or anti-polyoma T antigen mono-

clonal antibody PAb F4 (Pallas et al., 1986). As seen in Figure lA, PAb F4 precipitated solely polyoma T antigen, while PAb 421 immunoprecipitated only ~53. These results demonstrated that under these conditions, polyoma T antigen is unable to form a complex with p53 in insect cells. Similar results were observed in rat cells expressing polyoma large T antigen (Manfredi and Prives. unpublished data), suggesting that despite their similarity, SV40 and polyoma large T antigens differ in their interactions with the host p53 protein. Ceils that do not express SV40 T antigen generally produce insufficient quantities of wild-type p53 for brochemical analysis. However, substantial quantities of wild-type murine p53 were produced in insect cells infected with the vEV55p53 baculoviral vector. Many other proteins, in particular SV40 (Murphy et al., 1988) and polyoma (Rice et al., 1987) T antigens, expressed in these cells have been shown to be biologically active and to retain properties exhibited by their mammalian counterparts (for review, see Miller, 1988; Lucknow and Summers, 1988). This suggested that p53 might also be functional when isolated similarly from infected Sf27 cells. To assess their relative abundance and purity, proteins were purified by immunoaffinity chromatography procedures (Figure lB), yielding preparations in which the predominant protein was either SV40 T antigen (lane l), polyomaT antigen (lane 2) or p53

Murine ~53 Inhibits SV40 DNA Replication 381

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(lane 3). The quantities of either SV40 or polyoma T antigen were comparable to or greater than those obtained with other viral vectors. The yields of recombinant baculoViral p53 were 5-to lOO-fold greater than those obtained from other cell lines that we screened.

-h--

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Dpnl:

Murine p53 Blocks SV40 but Not Polyoma ori-DNA Replication in Vitro

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P53 (WI) Figure 2. Effect of Murine ~53 on SV40 and Polyoma on-DNA Replication In Vitro (A) Reaction mixtures lacking (upper and lower panels; lanes 1) or containing 1.2 ug of SV40 T antigen (lanes 2-7, upper panel) or 0.7 ug of polyoma T antigen (lanes 2-7, lower panel) and either HeLa extracts and SV40 on-DNA (upper panel) or FM3A extracts and polyoma oriDNA (lower panel) and other components as described in the text were incubated with no p53 (lanes 1 and 2) or 0.1 (lanes 3) 0.2 (lanes 4). 0.4 (lanes 5). 0.6 (lanes 6), or 0.8 (lanes 7) ug of murme ~53. After incubation for 3 hr, DNA was purified from reaction mixtures, linearized with Pvul, and incubated with (+) or without (-) Dpnl. DNA samples were then subjected to electrophoresis in 1% agarose gels and autoradiography. (6) At the end of the incubation, 5 ~1 akquots of each reaction mixture were acid-precipitated and counted by liquid scinitillation. dTMP (1.5 and 1.6 pmols) was incorporated into acid-insoluble material in the absence of SV40 T antigen and polyoma T antigen, respectively.

Our ability to generate substantial quantities of purified wild-type p53 from baculovirus-infected insect cells allowed us to test the effects of the host protein upon SV40 and polyoma ori-DNA replication in vitro. Both SV40 and polyoma T antigens isolated from insect ceils mediated the replication of plasmids bearing their respective origins in cell-free extracts prepared from human HeLa or mouse FM3A cells, respectively (Figures 2A and 28). The quantities of DNA synthesized in vitro relative to the amount of recombinant baculoviral T antigen added to the reaction were comparable to those reported for T antigens isolated from mammalian cells infected with other viral vectors (Li and Kelly, 1984; Stillman et al., 1985; Wobbe et al., 1985; Murakami et al., 1986a), as measured by either acid precipitation or digestion with Dpnl, a restriction enzyme that cleaves methylated (bacterially propagated) DNA but not partially or fully unmethylated DNA synthesized during the in vitro reaction. As shown in Figure 2A, the replication of the SV40 oriDNA plasmid was dramatically reduced as increasing quantities of p53 were added, such that at the highest concentration of ~53, the level of incorporation did not exceed that observed in the absence of T antigen. However, similar concentrations of p53 had little or no effect on polyoma ori-DNA replication in vitro. It should be noted that levels of SV40 DNA replication were normalized to those of polyoma DNA replication in vitro, which were generally lower, by adding less SV40 T antigen to the reaction mixtures. At maximal levels of SV40 DNA synthesis, obtained by using more SV40 T antigen, proportionally greater quantities of ~53 were also completely inhibitory. Taken together, these results strongly suggested that ~53 inhibited SV40 oriDNA replication in vitro by binding to SV40 T antigen, and failed to inhibit polyoma ori-DNA replication because it cannot form a complex with the polyoma T antigen. We and others (Murakami et al., 1986a; Wang and Prives, unpublished data) have observed that HeLa cell extracts, in the presence of polyoma T antigen, support low levels of replication of plasmids bearing the polyoma replication origin, to an extent of approximately 5% of that observed in mouse FM3A extracts. When polyoma T antigen-mediated polyoma ori-DNA replication in HeLa extracts was examined in the presence of concentrations of murine ~53 sufficient to inhibit SV40 DNA replication completely, no repression was observed (data not shown). This supports the conclusion that the differential effects of ~53 on SV40 and polyoma DNA replication in vitro were due to differences in the two large T antigens and not to differences between the mouse and human cell extracts used in the replication reactions. Previous studies have indicated that with the exception of the DNA polymerase a-primase complex, the factors involved in the in vitro

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p53-

Figure 3. Complex Formation between Purified p53 and T Antigen In wro lmmunopurlfied SV40 T antigen (1 5 [lanes 1, 3, and 41 or 0 [lanes 21 pg) was Incubated wth 0 (lanes 1). 0 25 (lanes 3), or 0.75 (lanes 2 and 4) wg of lmmunopurlfled murine p53 as described in text. ReactIons were lmmunopreclpitated wth either PAb 419 (uSV4OT) or PAb 421 (ap53) coupled to protein A Sepharose and the proteins analyzed by electrophoresls In 10% SDS-polyacrylamide gels and silver staining.

replication of SV40 and polyoma ori-DNA are sufficiently similar that they can substitute for each other (Murakami et al., 1986b). Therefore, the inhibitory effect of ~53 on SV40 DNA replication is most likely due to its specific interaction with the viral T antigen.

p53 Forms a Complex with SV40 T Antigen In Vitro, and Purified p53-T Antigen Complex Inhibits the Replication Function of Free SV40 T Antigen To show that ~53 and SV40 T antigen associate in vitro, we established conditions for studying complex formation between the two purified proteins. Previous experiments demonstrating their ceil-free association had utilized purified T antigen and crude extracts containing p53 (McCormick et al., 1981; Schmieg and Simmons, 1988). As shown in Figure 3, when SV40 T antigen was incubated with increasing amounts of ~53, proportionally increasing quantities of the ~53 protein were immunoprecipitated by anti-T antigen antibody PAb 419. By comparing the amount of protein immunoprecipitated by PAb 419 and PAb 421 at each concentration of ~53, we found that the majority of the host protein added appeared to be efficiently bound to SV40 T antigen. Surprisingly, under these conditions only a small portion of the SV40 T antigen was associated with ~53. The ratio of T antigen to p53 did not change appreciably when p53-T antigen complex formation was examined under replication conditions--that is, in the pres-

ence of HeLa extracts and other components of the replication reactions (data not shown). When related experiments were performed with purified polyoma T antigen and ~53, no complex formation was observed (data not shown). This result presented a paradox: as determined by coimmunoprecipitation experiments, only a small proportion (10%-250/o) of the T antigen forms a complex with ~53. The remaining 750/o-90% of the unbound T antigen would be more than sufficient to mediate the replication of substantial quantities of DNA under conditions where no p53 was present. In considering various explanations for this discrepancy, it occurred to us that this might provide insight into how T antigen functions during viral DNA replication. Zonal density gradient sedimentation analyses have shown that T antigen exists as both monomers and oligomers in infected and transformed cells (Bradley et al., 1982; Gidoni et al., 1982; Montenarh and Henning, 1983; Runzler et al., 1987). T antigen was also shown to form multimeric complexes consisting of several monomers of T antigen when bound to viral DNA (Myers et al., 1981; Mastrangelo et al., 1985; Dean et al., 1987b). T antigen oligomers may thus play an important role iln SV40 DNA replication, and their function may be inhibited when ~53 is bound to one or more of the T antigen monomers. If this is the case, then p53 bound to T antigen should inhibit the ability of unbound T antigen to replicate or&DNA to the same extent as free ~53. To test this, insect cells were coinfected with both the SV40 T antigen and ~53 recombinant baculoviruses, and to obtain a preparation in which all the ~53 was bound to T antigen, the complex was immunopurified using the T antigen-specific antibody PAb 419. Evidence that the majority (if not all) of the ~53 present in the preparation was bound to T #antigen was provided by their coimmunoprecipitation with PAb 419 and cosedimentation on sucrose gradients (data not shown). Furthermore, quantities of ~53 isolated in complex with T antigen were similar to those of free p!j3 purified from insect cells infected with the ~53 vector alone (Figure 4A). When we compared the abilities of free ~53 and the p53-T antigen complex to affect free T antigen-mediated replication of o/Y-DNA in vitro, both preparations inhibited DNA synthesis (Figure 48). The p53-T antigen complex was, in fact, somewhat more inhibitory. The simplest interpretation of this result is that T antigen molecules must interact with each other during DNA replication, and that ~53 inhibits this interaction. If this interaction involves T antigen multimers, then it may be that as few as one molecule of T antigen in complex wlith ~53 would inhibit multimer function, thus explaining why so little of the p53-T antigen complex is required for the complete inhibition of SV40 DNA synthesis in vitro. To understand further which functions of T antigen are affected by ~53, we determined the time during the reaction at which the host protein inhibited DNA replication.

~53 Inhibits the Early Stages of SV40 o&DNA Replication Earlier studies on the time course of SV40 DNA replication in vitro showed that prior to the incorporation of deox-

Murine p53 Inhibits SV40 DNA Replication 393

lnittation in Vitro

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Figure 4. Inhibition of SV40 on-DNA Replication

In Vitro by Purified Free p53 and p53-T Antigen Complex.

(A) Murine p53 complexed to SV40 T antigen was purified from Sf27 cells coinfected with vEV55SVT and vEV55p53 baculoviral vectors usmg antiSV40 PAb 419-protein A Sepharose. p53 free of T antigen was immunoaffinity-purified from insect cells infected with vEV55p53 alone using PAb 421-protein A Sepharose. p53 bound to T antigen (0.75 and 1.5 ug) (lanes 1 and 2) compared with similar quantities of free p53 (lanes 3 and 4) were analyzed by PAGE and silver staining. Lane M: protein size markers whose molecular weights in kilodaltons are indicated on the right. (B) Reaction mixtures containing 0.75 ug of SV40 T antigen were incubated for 3 hr at 37% with increasing amounts of free ~53 (0) or p53-T antigen (0). The amount of acid-precipitable radioactivity was determined by liquid scintillation.

ynucleotides (dNTPs) into progeny DNA, there is a delay that is most likely due to the assembly of a pre-synthesis complex (Wobbe et al., 1986; Fairman and Stillman, 1988; Wold and Kelly, 1988). It was of interest to determine which stages of DNA replication were affected by ~53. To examine this, the reaction components were incubated for 30 min in the absence of the dNTPs, a condition previously established as delineating the pre-synthesis period of DNA replication in vitro (Wobbe et al., 1986). When the dNTPs were then added to the reaction, the characteristic time lag was abolished, and DNA synthesis was detected at the first time point examined (Figure 5A). When the presynthesis incubation was carried out in the presence of ~53, no significant amount of synthesis above background was detected at all time points up to the termination of the reaction. These results demonstrated that p53 hinders SV40 T antigen during the pre-synthesis stage of DNA replication. When the pre-synthesis complex was allowed to form by incubation in the absence of dNTPs, followed by the simultaneous addition of both the dNTPs and ~53, only a very slight inhibition of DNA synthesis was observed after 15 min (Figure 58). Thisis in contrast to what had been observed during the same time period when ~53 had been present from the start of the reaction. The addition of p53 still later in the reaction, i.e., 10 min after the addition

of the dNTPs, lengthened the delay such that the inhibitory effect was not detected until approximately 1 hr after the onset of elongation (Figure 5C). As the rate of DNA synthesis in vitro has been previously shown to be on the order of 200 nucleotides per min (Wobbe et al., 1986), the completion of a round of synthesis of the 9600 bp plasmid used in these experiments would require less than 1 hr. This suggested that under these conditions, ~53 inhibited only the initial stages of DNA replication. We therefore were interested in examining the effects of ~53 upon those activities of T antigen that pertain to its role in initiation.

Binding of SV40 T Antigen Origin Is Inhibited by p53

to the Replication

The specific binding of T antigen to sequences within the viral origin is one of the initial functions of the viral protein. Mutations within this region that abolish T antigen binding also abolish DNA replication (Deb et al., 1986). Because we found that the early stages of replication were selectively inhibited by ~53, we examined its influence upon the binding of T antigen to DNA containing the viral origin of replication. Earlier reports had provided evidence that the p53-T antigen complex can bind specifically to a DNA fragment containing the entire regulatory region (Reich and Levine, 1982; Scheller et al., 1982). However, as this region contains all three T antigen binding sites, 1, 2, and

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time (min) Frgure 5. Trme Course of SV40 on-DNA Replicatron

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rn the Presence and the Absence of ~53

(A) Reaction mixtures lacking dNTPs and [a-sap] dTTP and contaimng 0 ag (0) or 1.2 frg (0,O) of SV40 T antigen were prerncubated for 30 min at 37% rn the presence (0) or absence (0) of ~53. dNTPs and [@P] dTTP were then added (trme = 0 min) and the incubation contrnued. The amount of acid-insoluble radioactivity in 5 nl aliquots was determmed at the indicated times. (6) Reaction mixtures as described above containing 1.2 pg of T antigen were preincubated for 30 min in the absence of ~53. At the time rndrcated by the arrow (time = 0) dNTPs and [a-szP] dTTP were added along with D buffer containing no protern (0) or containing ~53 (0) The reaction was contmued at 37°C for the Indicated times, after which the amount of acid-insoluble incorporation was determined. (C) Reactrons were preincubated as described above. Following the addition of dNTPs and [u-~*P] dTTP at trme = 0 min, D buffer (0) or D buffer contarning ~53 (0) was added to the reaction mixture at time = 10 min, as Indicated by the arrow. Aliquots were acid-precipitated at the rridrcated times to determrne the rncorporatron of [a-s2P] dTMP.

3, (Tjian, 1978), only one of which is within the core origin of replication (site 2), these experiments did not distinguish between any possible differential effects of ~53 upon the interaction of T antigen with these sites. Furthermore, they were performed under conditions that were subsequently shown not to be optimal for site 2 binding (Deb and Tegtmeyer, 1987; Borowiec and Hurwitz, 1988a). To characterize further the effects of ~53 on the binding of T antigen to these sites, fragments containing either intact sate 1 or site 2 (Strauss et al., 1987) were purified and each bound separately to T antigen under conditions that closely resembled those used in the DNA replication reactions, Le. in the presence of ATP, and incubation at 37% (Figure 6). Using a filter binding assay, it was observed that binding to site 1 was not affected by relative concentrations of ~53 that abolished the replication of ori-DNA in vitro. In contrast, the presence of similar quantities of ~53 significantly reduced binding to site 2. ~53 alone did not bind to either site 1 or site 2 under these conditions (data not shown). These observations were confirmed using an

alternative binding assay (McKay, 1981) in which these DNA fragments were bound to either free T antigen or p53-T antigen complex followed by immunoprecipitation with PAb 419. We observed that either free or complexed T antigen bound well to the site 1 fragment, while only the free T antigen bound efficiently to the site 2 fragment (unpublished data). The degree of inhibition of site 2 binding was dependent upon the ratio of p53 to T antigen, as had been observed with SV40 ori-DNA replication, but was never absolute, even at ~53 concentrations that completely inhibited ori-DNA replication in vitro. This suggested that other activities of T antigen may also be affected by ~53.

Helicase and DNA Unwinding Activities of T Antigen Are Markedly Reduced in the Presence of p53 The role of T antigen in the initiation of viral DNA replication requires not only its specific DNA binding, but also its ATP-dependent helicase activity. These two properties

Murine p53 Inhibits SV40 DNA Replication 305

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in Vttro

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p53 Inhibits SV40 T Antigen Binding to Site 2 but Not Site 1

Bmdmg reactions containing quantities of SV40 T antigen as indicated rn the absence (0) or presence (0) of 0.5 pg of ~53, and 15 ng of either 3zP-end-labeled DNA fragments contaimng site 1 or site 2 were incubated for 15 min at 37°C and filtered through 0.45 pm nitrocellulose filters and counted by liquid scintillation

are presumably involved in the ability of T antigen to unwind DNA at the replication origin (Dean et al., 1987a). We therefore examined the effects of ~53 on these activities. The helicase activity of T antigen was determined by incubating the purified viral protein with a labeled heteroduplex substrate consisting of a [32P]-end-labeled 31 nucleotide oligomer annealed to a complementary circular single stranded Ml3 DNA derivative in the presence of ATP (Table 1). T antigen displaced amounts of the labeled oligonucleotide comparable to previously published studies (Stahl et al., 1986; Dean et al., 1987a). However, in the presence of increasing quantities of ~53, a marked decrease in the amount of oligomer displaced from the heteroduplex was observed. ~53 itself exhibited virtually no helicase activity (data not shown). Because ~53 inhibited both the site 2 binding and helicase activity of T antigen, it was predicted that it should strongly repress the T antigen-mediated unwinding of oriDNA described by Dean et al. (1987a) and Wold and Kelly (1988). The ability of T antigen to unwind DNA was determined by incubating relaxed ori-DNA in the presence of E. coli single-stranded binding protein (SSB), calf thymus topoisomerase I, and ATP. This generated the appearance of a species of ori-DNA previously termed U-DNA (Dean et al., 1987a) that migrated in the vicinity of form I supercoiled DNA (Figure 7A). The specificity of the reaction was

Table 1. Effect of Murine ~53 on Helicase Activity of SV40 T Antigen P53 (vg)

Drsplaced

0.0 0.4 0.8

33.3 12 1 3.0

Oligomer

(fmols)

Helicase assays were performed as described in Experimental Procedures. All reactrons contained 0.4 ug of SV40 T antigen. Values represent the average of two separate experiments and were adjusted for quantities of oligomer displaced in the absence of T antigen

supported by the fact that incubation of similar reaction mixtures containing a plasmid (pAT1.53) that lacks the viral origin did not yield any U-form DNA (Figure 78). To examine the reaction products in more detail, U-DNA was gelpurified and then treated with topoisomerase I (Figure 7C). The appearance of substantial quantities of Form II DNA after such purification, as seen in Figure 7C, has been noted by others (Dean et al., 1987a). Topoisomerase I converted the T antigen-dependent U-DNA to relaxed Form II DNA, demonstrating that it was a topoisomer. To characterize the effects of ~53 on unwinding, T antigen was incubated with relaxed ori-DNA in the presence of E. coli single-stranded binding protein (SSB), topoisomerase I, ATP, and increasing quantities of purified ~53. As seen in Figure 7A, addition of ~53 inhibited the formation of T antigen-dependent U-DNA. Furthermore, p53-T antigen complex, prepared as in Figure 4, was both itself unable to unwind o&DNA and also inhibited the formation of U-DNA by free T antigen, much as it had inhibited the ability of free SV40 T antigen to mediate the replication of oriDNA in vitro. p53 itself showed no ability to unwind either relaxed o&containing or ori-lacking DNA (data not shown). Importantly, when ~53 was incubated with the purified reaction products, the quantities of U-DNA did not diminish appreciably (Figure 7D). Our data therefore strongly suggest that the reduction of binding to site 2, and the inhibition of helicase activity by p53 work in concert to block the ability of T antigen to unwind the DNA template, preventing the initiation of SV40 ori-DNA replication in vitro. They further support the idea that the unwinding activity of T antigen requires interactions between T antigen molecules within multimers that cannot occur when T antigen is bound to ~53.

A Tranforming Mutant ~53 Is Altered in Its Ability to Block SV40 DNA Replication In Vitro Recently it was shown that mutant but not wild-type forms of murine p53 cooperate with ras to transform primary cells in culture (Hinds et al., 1989). It was therefore of in-

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0 In a +

Activity of SV40 T Antigen

(A) Unwinding reaction mixtures containing pATSV0 (ori+) DNA, 0 ug (lane 1, 7, 8) or 0.75 ug of free SV40 T antigen (lanes 2-6) and 0 (lanes 1 and Z), 0.2 (lane 3) 0.4 (lane 4) ug of free ~53 or 0.4 (lanes 5 and 7) 0.8 (lanes 6 and 8) ug of p53-T antigen complex were Incubated for 2 hr at 37%. The products were subjected to electrophoresis in 1% agarose gels followed by Southern blotting and hybridization to a 32P-labeled pATSV0 probe. Positions of supercoiled (I), nicked circular (II), and linear (Ill) DNA as determined by DNA markers run in parallel lanes but not shown here are indicated at left. An example of such markers is seen in (B), lanes A and B. U refers to unwound DNA. (B) pAT153 (ori-) DNA was incubated in reaction mixtures containing 0 (lane 1) or 1.0 (lane 2) ug of T anbgen for 2 hr at 37°C. The reaction products were detected as described above. Lanes A and 6 contain: supercoiled (I), nicked circular (II), and linear (Ill) forms of pAT153 DNA. (C) Unwound (form U) DNA visualized in 1% agarose gels by ethidium bromide staining was excised from the gel and purified by electroelution. The isolated DNA was treated with 0 (-topo I) or 10 (+topo I) units of topoisomerase I for 1 hr at 37% under unwinding reaction conditions. The products were separated in 1% agarose and detected by Southern hybridization. The position of forms I, II, and Ill DNA was determined by size markers not shown. U indicates the position of unwound DNA. (D) Reaction mixtures containrng on+ DNA and T antigen as in (A) lane 2, were deproteinized and incubated for 1 hr at 37°C with 0 (-~53) or 1.0 (+p53) ug of ~53 under conditions identical to those in the unwinding reaction. The products were electrophoresed and detected as described in (A). Forms I, II, Ill, and U of pATSV0 DNA are indicated as determined by DNA markers not shown.

terest to compare the ability of a transforming and a nontransforming ~53 protein to affect T antigen-mediated replication of ori-DNA in vitro. One such mutant ~53, Ch53-7 (also termed p53cG; Bienz et al., 1984) containing a single amino acid difference at position 135 (ala to val) was used to transform primary rat cells in conjunction with ras. A cell line, termed clone 6, obtained in this fashion, expressed high levels of the mutant protein, ~53”~’ (Pinhasi-Kimhi et al., 1986). It was therefore possible to purify comparable quantities of ~53 from clone 6 cells and from insect cells infected with vEV55p53, which encodes wild-type ~53, ~53~‘~ (Figure 8A). When we compared the abilities of the mutant and the wild-type purified ~53 proteins to inhibit the replication of SV40 ori-DNA, a somewhat unusual result was obtained (Figure 8B). With low concentrations of either mutant or wild-type ~53, DNA replication was partly inhibited. However, as the concentrations of the two proteins were increased, their effect on DNA synthesis differed dramatically. Whereas wild-type ~53 inhibited T antigen-mediated DNA replication to proportionally greater extents, the amount of DNA replicated in the presence of greater quantities of ~53”~’ increased until it reached levels similar to those observed when no ~53 was present. This was observed reproduci-

bly with several different preparations. Thus, at concentrations at which wild-type ~53 was the most inhibitory, the mutant ~53 did not appreciably affect SV40 DNA replication. One possible interpretation of these results is that at higher concentrations ~53”~’ is unable to bind to T antigen. In fact, our preliminary results have indicated that ~53 from clone 6 cells bound to T antigen significantly less efficiently than did wild-type ~53 (data not shown). These results show that at least one transforming mutant ~53 is markedly different in its ability to affect the replication of SV40 ori-DNA mediated by large T antigen, when contrasted with the wild-type nontransforming ~53. Discussion The T antigens encoded by SV40 and polyoma share greater than 60% homology (Soeda et al., 1980; Tooze, 1981). Each is the sole viral protein required for viral DNA replication, and both exhibit very similar biochemical activities pertaining to this role. Each binds with high affinity to sites within DNA containing multiple copies of the consensus pentanucleotide 5’ GAlGGGC 3’ that are present within and adjacent to each viral replication origin (Tegtmeyer et al., 1983; Jones and Tjian, 1984; Cowie and Ka-

Murine p53 Inhibits SV40 DNA Replication

lnitiatlon In Vitro

307

B M

123456 60 -

P53 (KU

Figure 6. Inhibition of SV40 DNA Replication

by Mutant ~53”~’ Differs from Wild-Type ~53~‘~

(A) WT ~53~‘~ and mutant ~53”~’ were purified as described in Experimental Procedures. ~53~‘~ (2. 5, and 10 ~1) (lanes 1, 2, and 3) or ~53”~’ (lanes 4,5, and 6) was separated on 10% SDS-polyacrylamide and visualized by silver staining. The polypeptide migrating more rapidly than the full-length ~53”~’ shares at least one epitope with ~53, as determined by Western blotting. Its abundance, which varied with different preparations, did not correlate with the highly reproducibly effect of the mutant ~53. Lane M: protein size markers from top are 92.5 kd and 45 kd. (6) Reaction mixtures containing 0.75 lg of SV40 T antigen and the indicated amounts of ~53~‘~ (0) or ~53”~’ (0) were incubated for 3 hr at 37% The amount of acid-insoluble radioactivity was determined at the end of the reaction period. Analysis of replication products on agarose gels, as described in Figure 2, provided essentially identical results.

men, 1984, 1986; Scheller and Prives, 1985). In addition, each protein is both an ATPase and a helicase (Tjian and Robbins, 1979; Clertant et al., 1984; Stahl et al., 1986; Wobbe et al., 1987). However, the two viruses exhibit a remarkably specific and distinct tropism for cells that are permissive for their replication. Polyoma replicates in mouse but not human or monkey cells, while SV40 replicates in primate but not murine cells. The development of systems that replicate SV40 and polyoma ori-DNA in vitro has provided insight into host-range specificity. It was shown that species-specific differences in components that are either intrinsic to or copurify with the DNA polymerase a-primase complex provide the basis for papovavirus host-range permissiveness (Murakami et al., 1986a, 1986b). Our data suggest that permissiveness may be governed by additional factors. SV40 but not polyoma T antigen fails to replicate ori-DNA in the presenceof murine p53 in vitro. The lack of p53 binding to polyoma T antigen may represent, then, an evoluUonary consequence of the repressive nature of the murine host protein. If its T antigen were to bind ~53, polyoma might not have evolved as an efficiently replicating mouse papovavirus.

Our experiments are supported by a recent study showing that plasmids encoding murine but not human ~53, when cotransfected with SV40 ori-DNA plasmids into Cos cells, lead to reduced levels of replicated ori-DNA (BraithWaite et al., 1987). This suggests that although murine and human p53 both bind to SV40 T antigen, only the association with the former represses the replication function of T antigen in vivo. Because sufficiently large quantities of purified human or monkey p53 are not yet available, it has not been possible to compare directly the relative effects of added primate and murine ~53 SV40 on ori-DNA replication in vitro. The finding that polyoma o/&DNA replication was not affected by murine p53 in either mouse or human extracts provided evidence that those cellular factors involved in the replication of polyoma, as well as of SV40 oriDNA, are not targets of ~53. The number and identification of cellular factors involved in papovavirus DNA replication is not well defined, although several groups have extensively fractionated replication extracts from human cells (Wobbe et al., 1987; Fairman and Stillman, 1988; Wold and Kelly, 1988). One report has described experiments using purified compo-

Cf?ll 388

nents in which the helicase activity required for SV40 and polyoma ori-DNA replication was supplied apparently exclusively by the T antigens themselves because no other helicase activity could be identified (Wobbe et al., 1987). This is consistent with previous reports suggesting that SV40 is associated with replication forks (Stahl and Knippers, 1983; Tack and Proctor, 1987) and is required for elongation of growing chains (Stahl et al., 1985; Wiekowski et al., 1987). We have shown here that ~53 drastically reduced the helicase activity of T antigen. However, adding ~53 after DNA elongation had initiated did not appear to inhibit that round of replication; only the pre-synthesis stage of ori-DNA synthesis was affected by the host protein. To reconcile these seemingly conflicting observations, different explanations can be proposed. If T antigen is required for chain elongation, then its resistance to ~53 during that phase may be such that once elongation has begun, T antigen is no longer accessible to ~53. It was reported that free T antigen binds to DNA polymerase a in vitro (Smale and Tjian, 1986), but cannot do so when associated with murine ~53 (Gannon and Lane, 1987). This may reflect a competition between these two cellular proteins for T antigen such that if polymerase is bound first, p53-T antigen complex formation is blocked. It was also shown that one population of T antigen that is associated with replicating chromosomes is extremely tightly bound, as measured by its resistance to high salt concentrations (Stahl and Knippers, 1983). Tantigen in this state may be unable to interact with ~53. If, on the other hand, T antigen does not normally function in DNA chain elongation, unwinding of DNA at the replication fork may be supplied by a cellular helicase. This helicase activity would be present and operational in systems containing cruder but not more highly purified components. This is consistent with earlier studies that showed that tsA conditional mutants of T antigen function like “slow stop mutants” suggesting that the viral A gene protein is required only for initiation of new rounds of viral DNA replication in vivo (Tegtmeyer, 1972; Chou et al., 1974). Experiments to distinguish between these possibilities are in progress. Our experiments have also provided new information about the properties of SV40 T antigen. They have confirmed that T antigen interacts differently with binding sites 1 and 2 because addition of ~53 only affected its binding to the latter. In contrast to what we had observed with either the helicase or the unwinding assays, it was not possible to block binding to site 2 completely, even at concentrations of ~53 that had abolished SV40 or&DNA replication in vitro. This could be explained if T antigen binds specifically to DNA in both monomer and multimer form, as has been suggested by Mastrangelo et al. (1985). Even if the p53-T antigen complex affected the ability of T antigen multimers to bind, there should still be sufficient free T antigen in monomer form to bind site 2. Although both sites 1 and 2 contain several copies of the 5’ GA/GGGC 3’binding sequence, the arrangement of these motifs both in terms of spacing and neighboring sequences differs within these two sites and has been shown to affect their relative affinity for T antigen (DeLucia

et al., 1983; Ryder et al., 1985; Scheller and Prives, 1985). These differences might position the T antigen molecules on the DNA differently; hence, their interaction with one another at these sites may be fundamentally dissimilar. Alternately, as the affinity of T antigen for site 1 is significantly greater than for site 2, the presence of small quantities of T antigen in complex generated in vitro may have little effect on binding of free monomeric T antigen to this site. In the presence of ATP and other nucleotides, T antigen binding to site 2 is specifically augmented, presumably due to a nucleotide-dependent conformational alteration (Deb and Tegtmeyer, 1987; Borowiec and Hurwitz, 1988a). Consistent with this is the observation that T antigen forms multimeric complexes at the origin in an ATPdependent manner under conditions that support DNA unwinding and replication (Dean et al., 1987b). It should be noted that ~53 did not affect the ATPase activity of T antigen (unpublished data), suggesting that the host protein does not interfere with its binding to ATI? The presence of one or more molecules of ~53 in the ATPdependent T antigen multimers may inhibit the ability of these multimers to induce localized melting and structural changes within the replication origin as described by Borowiec and Hurwitz (1988b). Evidence that interactions between the T antigen molecules in these complexes is required for binding and/or unwinding was provided by our experiments showing that both free ~53 and T antigen-bound ~53 inhibited DNA synthesis and unwinding. The ability to form the p53-T antigen complex in vitro from purified components will enable us to characterize further the effects of ~53 on T antigen function. In contrast to our observation that increasing concentrations of wild-type murine ~53 led to proportionally decreasing levels of SV40 T antigen-mediated DNA synthesis in vitro, the mutant ~53”~’ from clone 6 cells exerted a markedly different effect. Higher but not lower quantities of ~53”~’ failed to inhibit SV40 DNA replication. This effect could best be explained if mutant p53 formed oligomers in a concentration-dependent fashion that were unable to interact with T antigen. Indeed, it was reported that ~53 in clone 6 cells is largely present as very high molecular weight, oligomeric forms, while in untransformed cells it is present predominantly in monomeric form (Kraiss et al., 1988). It is interesting that the ability of the ~53”~ from clone 6 cells to cotransform primary cells with ras was demonstrated utilizing a vector that generated high levels of ~53 (Hinds et al., 1989). In fact, the overexpression of other mutant ~53s under a strong promoter yielded a higher efficiency of transformation than those under a weaker promoter (Finlay et al., 1988). Therefore, there is an intriguing correlation between the transformation efficiency of high levels of ~53”~’ and its inability to inhibit SV40 DNA replication. It has been proposed, based on several lines of diverse evidence, that p53 may normally function as an anti-oncogene (reviewed by Green, 1989). Lack of functional ~53 would then lead to unregulated DNA replication and cell division. One mode by which SV40 transforms cells may be via the binding to and sequestering of cellular ~53, thus preventing its normal role

Murine ~53 Inhibits SV40 DNA Replication 389

Initiation in Vitro

in growth regulation. In a similar fashion, high concentrations of mutant p53 may also sequester wild-type p53 by dint of forming hetero-oligomers (Eliyahu et al., 1988). Furthermore, that polyoma large T antigen, as we have shown here, fails to bind ~53 may be related to the fact that unlike its SV40 counterpart, it does not, by itself, transform cells (Rassoulzadegan et al., 1982). In summary, we have, in these studies, identified a biochemical function of wildtype ~53, namely the inhibition of SV40 DNA replication. That the transforming mutant ~53”~’ from clone 6 cells did not block this process provides the basis for future examination of additional mutant ~53 proteins that have been shown to have oncogenic properties and may eventually provide insight into the cellular function of the host protein. Experimental

Procedures

Cells and Viruses Spodoptera frugiperda insect cells (Sf27 cells) and recombinant baculoviruses vEV55SVT (SV40 T antigen), vEV55p53 (murine ~53) and vEV5lLT (polyoma T antigen) were kindly provided by D. O’Reilly and L. Miller (Rice et al., 1987; O’Reilly and Miller, 1988). Sf27 cells were grown at 27°C in TC-100 medium (GIBCO) containing 10% heat inactivated fetal calf serum and 0.25% tryptose broth. Clone 6 rat cells expressing mutant ~53”~’ were obtamed from D. Michalovrtz and M. Oren. Purification of SV40 T Antigen, Polyoma T Antigen, and Murine ~53 Sf27 cells (2.5 x lo7 per 150 m m dish) were infected with recombinant vrruses and harvested 48 hr post infection. Cultures were washed twice in cold phosphate-buffered saline (1 m M Na2HP04, 10.5 m M KH2P04, 140 m M NaCI, 14 m M KCI [pH 6.21) extracted with 1.6 ml of lysis buffer (50 m M Tris-HCI [pH 8.01, 150 m M NaCI, 1% Nonidet P-40 [NP-401, 0.1% aprotinin, 10 nM benzimidine, 30 pglml of leupeptin, 1 Kg/ml of bacitracin, 10 pglml of us-macroglobulrn, 1 m M DTT, and 0.35 m M PMSF) and incubated on ice for 30 min. Lysates were centrifuged at 2000 rpm for 15 min followed by centrifugation at 20,000 rpm for 30 min. Clarified supernatants were loaded onto the appropriate monoclonal antibody column (PAb 419 cross-linked to protein A Sepharose for SV40 T antigen preparations that either contained or lacked ~53, PAb F4 coupled to anti-mouse IgG agarose beads for polyoma T antigen, and PAb 421 cross-linked to protein A Sepharose for ~53) and purified by immunoaffinity chromatography (Simanis and Lane, 1985; Murakami et al., 1986a). Peak fractions as determined by silver staining were pooled and dialyzed agarnst D buffer (10 m M HEPES IN-2-hydroxyethylpiperazine-N’-2-ethanesulfonic acid] [pH 7.51, 5 m M NaCI, 0.1 m M EDTA, 50% glycerol, 1 m M DTT). SV40 T antrgen, munne ~53, or T antigen-bound murine p53 were purified from 10 infected 150 m m plates using 1 ml columns containing approximately 75 ng of antibody. Preparations of polyoma T antigen required 30 infected 150 m m plates and were loaded onto antibody columns containing approxrmately 50 ng of PAb F4 antibody. ~53”~’ was extracted from 5 x 10s clone 6 cells with buffer A (10 m M Tris-HCI [pH 8.01, 150 m M NaCI, 2% NP-40, 1 m M DTT, 10 nM benzimrdine, 30 nglml of leupeptin, 1 nglml of bacitracin, 10 uglml of a,-macroglobulin, and 0.35 m M PMSF) and purified by immunoaffrnity protocols similar to those used for isolating p53 from insect cell extracts. In Vitro DNA Replication HeLa extracts were prepared accordrng to publrshed procedures (Wobbe et al., 1985). Preparation of FMBA extracts was similar to that used for HeLa cells with the following modification: after the cells were dounce-homogenized and the release of nuclei was observed, the lysate was immediately centrifuged at 20,000 rpm for 30 min. and the supernatant was stored m aliquots at -80%. Standard reaction mrxtures (50 ul) contarned 40 m M creatine phosphate (pH 7.7; di-Irrs salt); 7 m M MgCI,; 0.5 m M DTT 4 m M ATP; 200 FM each CTP UTP GTP;

100 nM each dATP dGTP dCTP; 20 nM [a-s’P] dTTP (2 x 104 cpmlpmol) and 100 nglml of creatine kinase. For SV40 ori-DNA replrcation. 350 ng of pSV3 DNA (the entire SV40 genome inserted at the BamHl site of pBR322), HeLa extract (300-400 ng of protein), and SV40 T antigen were added to the reaction, and the incubation was carried out at 37%. For polyoma on-DNA replication, 300 ng of pBE102 (A2 strain of polyoma, nucleotides 5022 to 1562, inserted into a pML vector; Kern et al., 1985) FM3A extract (300-400 ng of protein), and polyoma T antigen were added and the reaction was carried out at 33%. After the indicated times, incorporation of dTMP was determined by acid precipitation of reaction mixtures. To analyze the products, the reactions were terminated by the addition of SDS to 0.2% and EDTA to 15 mM, followed by proteinase K digestion (0.2 mglml) for 1 hr at 37%. The DNA was then extracted once with phenol, once with chloroform, and ethanol-precipitated. The samples were digested with the restriction endonuclease Pvul, in the presence or absence of Dpnl, and subjected to 1% agarose gel electrophoresis for 1 hr at 200V followed by autoradiography. In Vitro p53-T Antigen Binding Binding reactions contaming immunopurified SV40 T antigen and/or ~53 in 200 nl of buffer containing 10 m M Trrs (pH 8.0) 150 m M NaCI, and 1.5% NP-40 were Incubated for 30 min at 20%. Reaction mixtures were divided in half and immunopreciprtated with either PAb 419 or PAb 421 cross-linked to protein A Sepharose. Immune complexes were incubated on a rotating shaker for 3-16 hr at 4°C followed by washing four times in RIPA buffer (50 m M Tris [pH 7.2],150 m M NaCI, 1% NP-40, 1% sodium deoxycholate, and 0.1% sodium dodecyl sulfate [SDS]). Proteins were eluted from Sepharose matrices in electrophorests sample buffer and subjected to electrophoresis in 10% SDS-polyacrylamide gels followed by silver staining.

DNA Filter Binding Reaction mixtures (50 PI) contaming 40 m M creatine phosphate (di-Tris salt [pH 7.71) 7 m M MgCls. 0.5 m M DTT, 0.2 mglml of bovine serum albumin, 4 m M ATP 0.3 ug of pBR322, 15 ng of 5’ [sap]-labeled DNA fragment, SV40 T antigen and/or p53 where indicated were Incubated for 15 min at 37°C. Reaction mixtures were filtered through 0.45 frrn nitrocellulose filters presoaked in wash buffer (100 m M NaPO, [pH 7.01, 0.5 m M EDTA, 0.5 m M DTT), washed twice with the same buffer, dried, and counted. Helicase Assay Reactron mrxtures (15 @I) containing 25 m M Tris-HCI (pH 7.8) 5 m M DTT, 5 m M ATP 10 m M MgCIs, 15 ng of heteroduplex consisting of a 3zP-labeled 31 mer annealed to single-stranded MlSmpSSVO (Auborn et al., 1988) and SV40 T antigen in the presence or absence of ~53 were incubated for 60 min at 37%. The samples were then subjected to 10% PAGE and autoradiography to visualize the displaced oligomer that was excised from the gel and counted by liquid scintillation. DNA Unwinding Assay Reaction mixturers (30 nl) as described by Dean et al. (1987a) containmg 40 m M creatine phosphate (di-?-is salt [pH 7.71) 7 m M MgCIa, 1 m M DTT, 4 m M ATP 33 ug/ml of creatine kinase, 3 aglml of bovrne serum albumin (BSA), 0.2-0.4 ug of relaxed pATSV0 (SV40 nucleotides 5171 to 294 inserted into pAT153) or pAT153 DNA (Twigg and Sherratt, 1980). 4 U of calf thymus topoisomerase I (BRL). 0.6 ng of E. coli singlestranded binding protein (SSB; Pharmacia), HeLa extract (100 ug of protein), 0.75-1.0 ng of T antigen, and the indicated amounts of murine p53 or p53-T antigen complex were incubated for 2 hr at 37°C. The DNA was extracted with phenol and chloroform, ethanol-precipitated, and subjected to 1% agarose gel electrophoresis. The resulting gel was stained with 0.5 Kg/ml of ethidium bromide, transferred to nitrocellulose. and the reaction products were detected by hybridization with 32P-labeled pATSV0 DNA. The relaxed DNA substrate was prepared by incubating plasmid DNA for 1 hr at 37% with calf thymus topoisomerase I in 50 m M Tris (pH 7.5) 50 m M KCI, 10 m M MgCla, 0.5 m M DTT, 0.1 m M EDTA, and 30 nglml of BSA. The product was deproteinized, ethanol-precipitated, and resuspended in 10 m M Tris (pH 8.0), 1 m M EDTA.

Cell 390

Acknowledgments

Domam structure of the slmlan virus 40 core ongIn of repllcatlon Cell. BIoI. 6. 1663-1670

We are Indebted to D. O’Re~lly and L Miller for making recombinant baculoviruses encodlng SV40 T antigen and murme ~53 available to us prior to publtcatlon We are grateful to Ella Freulich for excellent techmcal assistance. J Manfredi and J. Manley are each also thanked for useful dlscusslons durmg the course of these studies and for crItIcal reading of this manuscript This work was suppported by grants CA 26905 and CA 33620 from the NatIonal lnstltutes of Health The costs of publication of this article were defrayed in part by the payment of page charges This article must therefore be hereby marked “adverffsement” In accordance with 18 USC. Section 1734 solely to indicate this fact

DeLucla, A L., Lewton, B. A , Tjlan. R.. and Tegtmeyer, F? (1983). Topography of simian wrus 40 A protein-DNA complexes arrangement of pentanucleotide Interaction sites at the orlgln of replication J Vlrol. 46, 143-150

Received October

12. 1988, revtsed February

15. 1989

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Harlow. E Crawford, L V., Plm. D. C.. and Wllllamson N M (1981) Monoclonal antIbodIes speclflc for slmlan virus 40 tumor antigens J Vlrol. 39. 861-869 Hinds. P. Fmlay. C.. and Levme. A. J (1989) Mutation IS required to activate the p53 gene for cooperation with the ras oncogene and transformation J Vlrol. 63. 739-746. Jenkins, J. R.. Rudge. K., and Currle. G A (1984). Cellular tmmortallzatlon by a cDNA clone encodmg the transformation-assocla’ied phosphoproteln ~53. Nature 372, 651-653 Jones, K. A.. and Tjlan. R. (1984). Essential contact residues wlthln SV40 large T antigen bIndIng sites I and II ldentlfled by alkylationInterference. Cell 36, 155-162 Kern, F G., Dailey, L and Baslllco, C (1985). Common regulatory elements control gene expression from polyoma early and late promoters In cells transformed by chlmenc plasmids Mol Cell Btol 5, 20702079. Kralss. S., Qualser, A., Oren, M.. and Montenarh. M (1988). Oligomerlzatlon of oncoproteln ~53. J. Vlrol. 62, 4737-4744 LI. J. J.. and Kelly, T J. (1984) Slmlan virus 40 DNA replication In vitro Proc Natl. Acad. SCI USA 81. 6973-6977 Lucknow, V. A., and Summers, M. D (1988). Trends In the development of baculovlrus expression vectors. Biotechnology 6. 47-55 Mastrangelo, I. A., Hough. P V. C., Wilson, V. G., Wall, J S HaInfIeld. J. F, and Tegtmeyer, P. (1985). Monomers through trlmers of large tumor antigen bmd in region I and monomers through tetramers bind In region II of slmlan virus 40 ongIn of replication DNA as stable structures In solution. Proc. Nat1 Acad. SCI. USA 82. 3626-3630 McCormick. F., Clark, R Harlow, E., and Tllan. R (1981). SV40 T ant,. gen binds specifically to a cellular 53 K protein ,n wtro Nalure 292. 63-65 McKay, R. (1981). Bmdlng of a slmlan virus 40 T antigen related protein to DNA J. Mol. Blot. 745. 471-488 Mercer. W. E , Nelson, D., DeLeo, A B., Old, L J and Baserga. R (1982) MIcroInjectIon of monoclonal antibody to protein ~5:) lnhiblts serum-Induced DNA synthesis In 3T3 cells. Proc Nat1 Acad SCI USA 79, 6309-6312 Miller, L. K. (1988). Baculovlruses Rev Microbial. 42, 177-179

as gene expression

vectors

Annil.

Murine ~53 Inhibits SV40 DNA Replication 391

Initiation in Vitro

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Cell 392

Weld. M. S.. LI, J J.. and Kelly, T. J. (1967). Initiation of simian virus 40 DNA replrcatron in vitro: large-tumor-antigen- and origin-dependent unwmdrng of the template. Proc. Natl. Acad. Sci. USA 84. 3643-3647 Wolf, D.. and Rotter, V. (1985). Mafor deletions in the gene encodrng the p53 tumor antrgen cause lack of p53 expression rn HL-60 cells Proc. Natl Acad SCI. USA 82, 790-794 Zajdel. M. E. El, and Blarr, G. E. (1988). The intracellular distribution of the transformation-assocrated protein p53 in adenovirus-transformed rodent cells. Oncogene 2, 579-584. Note Added in Proof In contrast to murine ~53, comparable quantities of wild-type human ~53 purified from Insect cells did not appreciably exhibit SV40 w-DNA replrcatron In vrtro