Temperature-sensitive mutants of adenovirus single-stranded DNA-binding protein. Inability to support DNA replication is associated with an altered DNA-binding activity of the protein.

The adenovirus single-stranded DNA-binding protein (DBP) is an essential factor in viral DNA replication. Three temperature-sensitive (ts) adenoviruses (Ad2+ND1ts23, Ad2ts111A, and Ad5ts125) are known to have single amino acid substitutions in their DBPs that result in defective DNA replication at the nonpermissive temperature. To elucidate the mechanism(s) involved in the ts phenotype, we purified the three mutant DBPs and studied their DNA-binding properties and their ability to support DNA replication in an in vitro system. The results confirm that the three ts DBPs were incapable of supporting DNA replication at the nonpermissive temperature (40 degrees C). The defect was found at both the initiation and elongation steps of DNA replication. The 2-fold stimulation of pTP.dCMP formation by the DBP was lost by prior heating of the ts DBPs. The pronounced effect of the DBP on the early elongation process was severely diminished, but not abolished, by prior heating to 40 degrees C. The functional change at 40 degrees C was irreversible, as the ts DBPs preincubated at 40 degrees C were no longer active when assayed at 30 degrees C. Upon heating to 40 degrees C, all three ts DBPs lost their ability to bind to oligonucleotides, although they still retained some binding activity for large single-stranded DNAs such as M13 DNA. Thus, the inability of these three ts DBPs to support DNA replication is attributable to their altered DNA-binding properties.

The adenovirus single-stranded DNA-binding protein (DBP) is an essential factor in viral DNA replication. Three temperature-sensitive (ts) adenoviruses (Ad2+NDlts23, Ad2tsll l A , and Ad6tsl26) are known to have single amino acid substitutions in their DBPs that result in defective DNA replication at the nonpermissive temperature. To elucidate the mechanism(s) involved in the ts phenotype, we purified the three mutant DBPs and studied their DNA-binding properties and their ability to support DNA replication in an in vitro system. The results confirm that the three ts DBPs were incapable of supporting DNA replication at the nonpermissive temperature (40 "C). The defect was found at both the initiation and elongation steps of DNA replication. The 2-fold stimulation of pTP*dCMP formation by the DBP was lost by prior heating of the ts DBPs. The pronounced effect of the DBP on the early elongation process was severely diminished, but not abolished, by prior heating to 40 "C. The functional change at 40 OC was irreversible, as the ts DBPs preincubated at 40 "C were no longer active when assayed at 30 "C. Upon heating to 40 "C, all three ts DBPs lost their ability to bind to oligonucleotides, although they still retained some binding activity for large single-stranded DNAs such as M13 DNA. Thus, the inability of these tbree ts DBPs to support DNA replication is attributable to their altered DNA-binding properties.
The adenovirus (Ad)' single-stranded (ss) DNA-binding protein (DBP), a 72-kDa phosphoprotein encoded in the E2A region of viral DNA, is expressed in large quantities during * This work was supported by National Institutes of Health Grant AI-17654, and by the American Lebanese and Syrian Associated Charities. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
8 To whom correspondence should be addressed.
The abbreviations used are: Ad, adenovirus; ss, single-stranded; ds, double-stranded; DBP, 72-kDa adenovirus ssDNA-binding protein; ts, temperature-sensitive; DNA-prot, adenovirus DNA with 55-kDa terminal protein covalently linked to each 5' end; pTP, 80-kDa precursor to terminal protein; Adpol, adenovirus DNA polymerase; V pTP and V pol, crude extracts prepared from HeLa cells infected with recombinant vaccinia viruses expressing Ad pTP and pol, respectively; NF, HeLa cell nuclear factors involved in adenovirus DNA replication; 2-ME, 2-mercaptoethanol; BSA, bovine serum albumin; HEPES, 4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid; DTT, dithiothreitol; SDS, sodium dodecyl sulfate; PAGE, polyacrylamide gel electrophoresis. both the early and late phases of virus infection. The DBP was originally characterized by its preferential binding to ssDNA (1) but was subsequently found to bind to the ends of double-stranded (ds) DNA (2,3) and to RNA (4,5). The protein performs multiple roles in various aspects of viral infection, but its major function is support of viral DNA replication.
Adenovirus DNA replication has been studied extensively since the first report on the development of an in vitro DNA replication system (6). At least six proteins have been found to be involved in the synthesis of full-length genomic DNA (reviewed in . Three of these are virally encoded proteins that are essential for DNA replication; the 140-kDa adenovirus DNA polymerase (Adpol), the 80-kDa precursor to the terminal protein (pTP), and the DBP. The pTP is essential for the protein-primed DNA replication mechanism (lo), in which the P-hydroxyl group of a serine residue (11) in the pTP serves as a primer (12) to which the first nucleotide, dCMP, is added by Adpol. The Adpol and pTP are tightly associated and are usually isolated from infected cells as a stoichiometric complex (13,14). The DBP is thought to play two roles in viral DNA synthesis: as a general ssDNAbinding protein that protects the displaced strand from nuclease attack (15) while separating it from the replicating template strand, and as an auxiliary factor for Adpol, enhancing processivity of the polymerase and facilitating DNA chain elongation (16,17). It has been inferred that the DBP specifically interacts with the Adpol in the presence or even absence of template DNA (17), although the existence of a stable Adpol. DBP complex has never been demonstrated.
In addition to viral proteins, at least three host cell nuclear factors (NFI, NFII, and NFIII) have been shown to be involved in viral DNA replication (18)(19)(20). NFI and NFIII bind to specific DNA sequences at the origin of DNA replication and stimulate the initiation process (21). NFII, a type I topoisomerase, is believed to be required for removing topological constraint, thereby permitting replication of genomelength DNA (19).
Genetic evidence for the requirement of DBP in DNA replication has accumulated from studies on conditionally lethal adenoviruses that have a mutation in the DBP (22-26). Ad5ts125 (27), which is identical to Ad5ts107 (28), is the prototype DBP mutant used in many studies, and has a Proto-Ser substitution at amino acid 413 of its DBP (28). Only a limited number of studies have used Ad2+NDlts23 or Ad2tslllA (29,30). These two viruses have single amino acid substitutions in their DBPs that are located in close proximity: Ad2tslllA has a Gly-to-Val change at amino acid 280 (30), while Ad2+NDlts23 has a Leu-to-Phe change at 282 16178 Temperature-sensitive Adenovirus DNA-binding Proteins 16179 (31). Interestingly, the region surrounding these two mutations has been proposed (32) to form a putative metal-binding domain known as a "zinc finger" (33). All three of the ts DBP mutants have been shown to be fully functional at the permissive temperature but defective at the nonpermissive temperature for supporting DNA replication in vivo (22)(23)(24) and in vitro (16,25,26,30). Efforts to find a change in the DNA-binding properties of these ts DBP mutants linked to their inability to support DNA replication at the nonpermissive temperature have produced inconclusive results. The first such study (34) demonstrated that the Ad5ts125 DBP was eluted from an ssDNAcellulose column at a lower temperature (20 "C) than was needed to elute wild-type DBP (40 "C), indicating that the mutant protein is defective in DNA-binding at elevated temperatures. This thermal elution, however, was carried out in the presence of 250 mM NaC1, a salt concentration inhibitory for DNA replication in vitro. A subsequent study (35) showed that the Ad5ts107 DBP was capable of binding to ssDNA, even at the nonpermissive temperature, if the binding was examined at salt concentrations comparable to that used for in vitro replication (20 mM NaCl). Similar results were recently obtained for the Ad2tslllA DBP (36), reinforcing the hypothesis that the defect for ts DBPs in DNA replication does not result from impaired DNA-binding activity, but may be due to an altered DBP .Adpol interaction. A puzzling finding has been reported for the Ad2+NDlts23 DBP (30): this mutant DBP was shown to bind to ssDNA very poorly at 4 "C but was fully functional in DNA replication at the permissive temperature (30 "C). Further study is clearly needed to explain this paradoxical finding.
Here, we report on studies using purified ts mutant DBPs (Ad2+NDlts23, Ad2tslllA and Ad5ts125) to examine a possible linkage between their ability to support DNA replication and their DNA-binding properties at the permissive and nonpermissive temperatures. Various assay systems were used to study slightly different aspects of DNA binding. Our results demonstrate that the DNA-binding properties of all three mutant DBPs change when they are heated to the nonpermissive temperature. The changes observed are quite obvious in some assays but equivocal in others. We believe that the defect of the ts DBPs in DNA replication can be ascribed to an alteration in their DNA-binding activity. DNAs-Synthetic oligonucleotides of various lengths (8,16,24,30, 40, and 84 nucleotides) were selected from our DNA primer collection produced by a DNA synthesizer (Model 380B, Applied Biosystems, Inc.) according to the following criteria: (a) no apparent palindromic sequence structure, and ( b ) no apparent bias in nucleotide content.
Purification of DBP-The DBPs were prepared from 2-liter suspension cultures of KB cells (4 X 10' cells/ml) infected with each virus at a multiplicity of 10, except for Ad2'NDlts23 which was used at a multiplicity of 3. Cells infected with wild-type virus were incubated at 37 "C and harvested at 50-h postinfection. Cells infected with ts viruses were incubated at 32.5 "C for 72-96 h. Purification of DBP was carried out according to the method of Schechter et al. (40) with two modifications: (a) DBP was eluted from an ssDNA-cellulose column with a linear rather than a step NaCl gradient; ( b ) 1 mM EDTA, 2 mM 2-mercaptoethanol(2-ME) and 0.2 mM phenylmethylsulfonyl fluoride were included in all buffers to protect ssDNAcellulose from nuclease attack and to stabilize proteins. DBP-containing fractions from the ssDNA-cellulose column were pooled and concentrated by vacuum dialysis against TEM buffer (10 mM Tris-HCl, pH 8.0; 2 mM 2-ME; and 1 mM EDTA) containing 0.5 M NaCl. The concentrated solution was further dialyzed against TEM containing 10% glycerol; insoluble materials (mostly aggregated DBP) were removed by centrifugation at 15,000 X g for 20 min. The supernatant (0.6-2.0 mg of protein/ml) was aliquoted in small quantities, quickly frozen in liquid nitrogen, and stored at -80 "C. Unless stated otherwise, DBPs were diluted to 100 pg/ml in TEM containing 20 mM NaCl, 10% glycerol, and 200 pg/ml bovine serum albumin (BSA) at 4 "C, and used within 2-3 weeks. Protein concentration was determined using the Bio-Rad protein assay dye reagent with BSA as the standard.
DNA Replication Assay-Adenovirus specific DNA replication was assayed in vitro by detecting the preferential incorporation of [3'P] dCMP into the origin containing terminal restriction fragments of Ad DNA-terminal protein complex (DNA-prot). Adenovirus DNAprot was prepared (41) from CsC1-purified Ad5dl301 virions (42) by equilibrium density gradient centrifugation in 4 M guanidine HCI, 2.7 M CsC1, 10 mM Tris-HC1, pH 8.0, 1 mM EDTA, and 1 mM phenylmethylsulfonyl fluoride. The DNA-prot was digested with XhoI (3 units per 1 pg of DNA-prot) prior to the reaction. Recombinant vaccinia viruses expressing Adpol and pTP were used to infect HeLa cells, and cytoplasmic extracts (designated as V pol and V pTP, respectively) were prepared as described by Stunnenberg et al. (37). A nuclear extract containing host cell factors was prepared from uninfected HeLa cells (6) and passed through a DEAE-cellulose column as described previously (43). Reactions were set up on ice by mixing 15 pl of a solution containing the indicated amounts of DBP with 10 pl of a solution containing other replication components, such that the final reaction (25 pl) contained 35 ng of XhoI digested Ad DNA-prot, 0.5 pl of V pol, 0.5 pl of V pTP, 0.15 p1 of uninfected HeLa cell nuclear extract, 0.8 pg of creatine kinase (240 units/mg), 7.5 mM phosphocreatine, 50 mM HEPES/KOH, pH 7.5,5 mM MgCl,, 2 mM dithiothreitol (DTT), 2 mM ATP, 60 p~ aphidicolin, 17 p~ each of dATP, dGTP, and dTTP, and 1 p~ [~y-~'PldCTp (100 Ci/ mmol). After incubation at 30 or 40 "C for the indicated periods of time, reaction products were precipitated by the addition of 12 pl of 8 M ammonium acetate, 3 pl of sonicated salmon testes DNA (0.5 mg/ml), and 100 p1 of ethanol. Pellets were dried and dissolved in 20 mM Tris-HC1, pH 8.0, 5 mM EDTA, 0.5% SDS, 10% glycerol, and 0.02% bromphenol blue, and then electrophoresed through a 1% agarose gel in TAE buffer (40 mM Tris-acetate, 20 mM sodium acetate, and 2 mM EDTA, pH 7.2) containing 0.1% SDS. Gels were partially dehydrated and autoradiographed at -80 "C using Kodak XAR film and Cronex Lightning-Plus intensifying screens.
Initiation and Partial Elongation Assays-Formation of initiation complexes (pTP.dCMP) was assayed by incubating the specified amounts of DBP with 50 ng of DNA-prot, 1 pl of V pol, 1 pl of V pTP, 0.3 pl of uninfected HeLa cell nuclear extract, 0.8 pg of creatine kinase, 40 mM HEPES/KOH, pH 7.5, 7 mM MgCI2, 1 mM DTT, 3 mM ATP, 7.5 mM phosphocreatine, 100 p M aphidicolin, 40 p M dideoxy-ATP, and 0.5 p~ [a-"PIdCTP (200 Ci/mmol) in a total volume of 25 pl (39). The formation of the partial elongation product (the first 26 nucleotides linked to pTP) was assayed with the same reaction mixture except that dideoxy-ATP was replaced with 40 p~ each of dATP, dTTP, and dideoxy-GTP. After incubation at 30 or 40 "C, reactions were stopped by the addition of 25 pl of a solution containing 200 mM sodium pyrophosphate and 50 mM EDTA followed by precipitation with 50 p1 of 50% trichloroacetic acid. Pellets were washed once with 5% trichloroacetic acid and dissolved in 40 pl of a loading solution consisting of 50 mM Tris-HCI, pH 6.8, 1% SDS, 1% 2-ME, 10% glycerol, and 0.02% bromphenol blue. The pH was neutralized by adding 1-2 pl of 1 M Tris base. Products were separated by SDS-PAGE (44) and visualized by autoradiography.
Synthetic Template Assay-DBP has been shown to specifically stimulate the synthesis of poly(dA) by Adpol with an oligo(dA): poly(dT) template (45). This stimulation was measured with 1 pg of DBP, 0.4 pl of V pol, 165 ng of poly(dT), 165 ng of oligo(dA), 50 mM Tris-HCI, pH 8.0, 8 mM MgCl2, 4 mM DTT, 200 pg/ml BSA, 3 mM ATP, and 8 p M [a-"PIdATP (125 mCi/mmol) in a final volume of 50 pl. DBPs were used without any heat treatment or after heating at 30 or 40 "C for 30 min. Reactions were carried out a t 30 or 40 "C for 30 min, and terminated by the addition of 950 pl of 5% trichloroacetic acid followed by filtration through HAWP filters. Incorporation of radioactive label into acid insoluble materials was measured by liquid scintillation counting.
Column Binding Assay-Details of the procedure have been described previously (46). In brief, wild-type ['HIDBP was made in KB cells with [3H]leucine, and purified through an ssDNA-cellulose column (47). The ts DBPs were labeled in vivo with ['"Plorthophosphate (0.3 mCi/ml) after infection of human 293.1 cells (1.5 X 10' cells) with the ts adenoviruses. For some experiments, whole cell extracts were prepared by sonication as previously described (47). The extracts were mixed with the purified wild-type [3H]DBP (approximately 5 X lo5 cpm) and loaded on ssDNA-cellulose columns (2-ml bed volume).
The columns were washed thoroughly with buffer A (10 mM Tris-HCI, pH 7.4,l mM EDTA, 2 mM 2-ME, and 10% glycerol) containing 0.1 M NaCl and then eluted with a 0.1-0.85 M linear NaCl gradient in buffer A. For studying the effects of heating, the "P-labeled ts DBPs were partially purified by ssDNA-cellulose prior to the column assay. The R2P-labeled extracts from Ad2tslllA-and Ad5ts125infected cells were loaded on ssDNA-cellulose columns, and the fractions between 0.30 and 0.85 M NaCl were collected. For Ad2+NDlts23, fractions between 0.23 and 0.65 M NaCl were collected instead. BSA was added as a carrier protein to the pooled fractions a t 200 pg/ml, and the solutions were dialyzed against TEM buffer containing 20 mM NaCl and 10% glycerol. This partially purified ts ["'PIDBP was mixed with the purified wild-type ['HIDBP (5 X lo5 cpm), and incubated at 40 "C for 30 min. The samples were centrifuged at 15,000 X g for 10 min, and the supernatants were loaded onto an ssDNA-cellulose or an ssDNA-agarose column (both 2 ml) followed by elution with an appropriate linear NaCl gradient. The elution profile of wild-type DBP was determined by liquid scintillation counting of 'H radioactivity. To detect "P-labeled ts DBPs, each fraction was immunoprecipitated (46) with a monoclonal antibody against DBP (B6 antibody in Ref. 48) and subjected to electrophoresis in SDS-containing polyacrylamide gels. Gels were dried and exposed to Kodak XAR film with an intensifying screen. Under these conditions, only "P radioactivity is detected, which allowed us to quantify ts DBPs by measuring the intensity of 72-kDa bands using a Hoefer GS-300 scanning densitometer.
Filter Binding Assay-To reduce nonspecific binding of ssDNA, nitrocellulose filters (HAWP) were treated with 0.4 M KOH for 30 min before they were used (2). Various amounts of DBPs, either unheated or heated at 40 "C for 30 min, were mixed with 20 ng of 'Hlabeled M13 DNA in 20 pl of TEM buffer containing 20 mM NaCI, 10% glycerol, and 200 pg/ml BSA. After overnight incubation at 4 "C, the mixtures were diluted 200-fold with TEM buffer containing 20 mM NaCl and passed through the KOH-treated filters. The DBP. DNA complexes retained on filters were washed three times with 2 ml of TEM containing 20 mM NaCI. Filters were dried and quantitated by liquid scintillation counting.
Gel Mobility Shift Assays-Various amounts of DBPs, either unheated or heated at 40 "C for 30 min, were mixed with a constant amount of R'P-labeled oligonucleotide or M13 DNA in 20 pl of a solution consisting of 10 mM Tris-HCI, pH 8.0, 20 mM NaCI, 1 mM EDTA, 2 mM 2-ME, 200 pg/ml BSA, and 10% glycerol. The mixtures were kept on ice for at least 30 min and analyzed at 4 "C by agarose gel electrophoresis (1% agarose for oligonucleotides and 0.5% for M13 DNA) using TBE buffer (45 mM Tris-borate, 1 mM EDTA, pH 8.3). Gels were partially dehydrated by being squashed on charged nylon membranes (BioTrace RP), and the gels and membranes autoradiographed together to locate DNA. For Western blot analysis, the gels and nylon membranes were briefly soaked in 3 M NaCl and 0.3 M sodium citrate, pH 7.0, and proteins and DNAs were further blotted (49) using the same buffer. To detect the DBPs, membranes were immunostained with an anti-DBP monoclonal antibody (B6) and an anti-mouse IgG HRP conjugate (Bio-Rad) according to the manufacturer's protocol.

RESULTS
Purification of DBPs-The DBPs of wild-type adenovirus (Ad5d1301), Ad2+NDlts23, AdZtslllA, and Ad5ts125 were purified to 90-9596 homogeneity, based on densitometer analysis of 72-kDa bands on SDS-PAGE (Fig. 1). All preparations contained a few extra bands in the 38-46-kDa region, some of which are presumably degradation products of the 72-kDa DBP (47). During purification, each DBP had a slightly different elution peak in ssDNA-cellulose chromatography: the wild-type DBP eluted at 0.60 M NaCl; Ad2+NDlts23 at 0.47 M; Ad2tslllA at 0.56 M; and Ad5ts125 at 0.55 M. We subsequently realized that comparisons based on NaCl concentrations were inaccurate because the elution peak for the same DBP varied significantly across column runs depending on the age of the column. To correct for this problem, we carried out an analytical column binding assay in which "P-labeled mutant DBPs were chromatographed together with wild-type ['HIDBP (Fig. 2). The results indicated that both Ad2tslllA and Ad5ts125 DBPs have a wildtype DNA-binding affinity, while the Ad2+NDlts23 DBP has a slightly decreased affinity.
Adenovirus DNA Replication in Vitro-Purified DBPs were tested in vitro for their ability to support adenovirus DNA replication. Although our assay system contained crude cell extracts, the system was free from possible contamination with DBP because Adpol and pTP were not prepared from adenovirus infected cells, but rather from cells infected with recombinant vaccinia viruses expressing Adpol and pTP (37).
The template DNA-prot was digested with XhoI prior to the reaction, so that successful replication should result in preferential incorporation of ["PIdCMP into the terminal fragments (XhoI B and C) which contain the DNA replication origins. As shown in Fig. 3  Wild-type and three ts DBPs (1.5 pg) were tested for their ability to support adenovirus DNA replication. Reactions were carried out either at 30 "C for 120 min or at 40 "C for 60 min. The DBPs were preincubated for 30 min at the reaction temperatures. The template DNA-prot was digested with XhoI prior to the reaction, so that successful replication should result in the incorporation of ['"PIdCMP into the origin containing end fragments ( B and C ) . Additional slower-migrating bands (B-RZand C-RZ) are replicative intermediates in which one of the parental strands is partially displaced by the new DNA but still connected to the template strand. Also labeled are faster-migrating single-stranded B and C fragments (ssB and ssC) that represent released, replicated strands. 40 "C, whereas the three mutant DBPs were functional at 30 "C (the permissive temperature) but not at 40 "C (the nonpermissive temperature), confirming their ts phenotype.
The functional change at 40 "C was an irreversible process: once the ts DBPs were heated to 40 "C, they were no longer functional even if chilled on ice for an extended period of time and then assayed at 30 "C (data not shown). This characteristic enabled us to study the heat-inactivation kinetics of the DBPs. At 40 "C, Ad5ts125 DBP was inactivated most rapidly, followed by Ad2+NDlts23 and Ad2tslllA (Figs. 4 and 5). Little inactivation was observed with wild-type DBP. A mixture of wild-type DBP and each ts mutant, after being heated at 40 "C, had replicative activity comparable to that of the wild-type DBP alone, indicating that the inactivated ts DBPs were not inhibitory to active DBP. It should be noted that when Ad5ts125 DBP lost its ability to support specific DNA synthesis, a smear of small 32P-labeled DNAs concomitantly appeared at the bottom of gel (Fig. 4). We do not know the origin of this smear. Similar smear bands also appeared with the heated Ad2tslllA and Ad2+NDlts23 DBPs, but they were seen only on an overexposed autoradiogram.
Initiation and Partial Elongation Assays-The in vitro assay described above can be modified to further investigate early events in adenovirus DNA replication. The initiation step is assayed by measuring the formation of the 80-kDa pTP. dCMP complex. An early elongation step is studied by substituting dGTP with dideoxy-GTP, which terminates DNA synthesis at the 26th nucleotide (pTP.26-mer, 88-kDa). The results of the initiation and partial elongation assays are shown in Fig. 6. Although the DBP was not essential for initiation, both wild-type and ts DBPs stimulated the formation of pTP -dCMP approximately 2-fold. This stimulatory activity was not seen when the ts DBPs were heated at 40 "C prior to the assay ( Fig. 6 and Table I). In contrast to the minor effect on the initiation reaction, the DBPs had a pronounced stimulatory effect on the early elongation process. Stimulatory activity of the ts DBPs for elongation was severely diminished, but not abolished, by prior heating to 40 "C ( Fig. 6 and Table I). The effect of heating was greater for Ad5ts125 DBP than for the other two ts DBPs, and the 88-kDa band produced with the heated Ad5ts125 DBP was not only fainter but also more diffuse than the other 88-kDa bands, indicating a possible heterogeneity in the initiation or termination sites.
Synthetic Template Assay-The elongation process was studied more closely using a synthetic template assay. This assay consists of only three components: Adpol, DBP, and oligo(dA):poly(dT) as a template. Neither pTP nor any of the host cell nuclear factors is required. In this system, the DBP specifically stimulates the synthesis of poly(dA) by Adpol in a dose-dependent fashion (17). As shown in Fig. 7, all three ts DBPs were capable of stimulating DNA synthesis at 30 "C, but their activity was abolished after incubation at 40 "C for 30 min. However, if the ts DBPs were not heated to 40 "C prior to the reaction, they remained active when assayed at 40 "C, suggesting that the ts DBPs bound to template DNA (together with Adpol) may be more resistant to heat-inactivation than those existing free in solution.
Column Biding Assays-The DNA replication defect of ts DBPs might result from changes in their DNA-binding properties. Several DNA-binding assays were used to test this possibility. We first used the column binding assay, because it gives us a rough estimate of DNA-binding affinity. Mixtures of ts ["PIDBP and wild-type [3H]DBPs were heated at 40 "C for 30 min, and then chromatographed on an ssDNA-cellulose column at 4 "C. Elution profiles are shown in Fig. 8. Comparison with Fig. 2 revealed little difference between the two elution profiles, indicating that the three ts DBPs were capable of binding to ssDNA as tightly as wild-type DBP after heating at the nonpermissive temperature. Similar results were obtained when another set of column assays were carried out at 40 "C using an ssDNA-agarose column (data not shown). At this temperature, ssDNA-cellulose could not be used because a substantial amount of ssDNA dissociated from the cellulose matrix.
Filter Binding Assuy-In the column assay described above, DNA-binding was tested under conditions where there was a large excess of DNA over the DBP. Under such conditions, especially at low salt concentrations, the ssDNA-cellulose may act not only as an affinity column but also as an ionexchange resin. Therefore, we next used a filter binding assay in which DNA binding can be studied over a wide range of DBP/DNA ratios in a low salt buffer (20 mM NaC1). The DBPs, unheated or heated at 40 "C, were mixed with 3Hlabeled M13 DNA and filtered through nitrocellulose filters (Fig. 9). Under the conditions used, the DBP and DBP.DNA complex were trapped on the filters, whereas the free DNA readily passed through. Nearly stoichiometric binding was observed with the unheated ts DBPs as well as with the wildtype protein. The ability of ts DBPs to bind to DNA was diminished by heating to 40 "C, but at saturating levels the heated proteins were still capable of retaining as much as 70% of the input DNA on the filters. Gel Mobility Shift Assay with Oligonucleotides-The ability The amounts of initiation product (80 kDa) and partial elongation product (88 kDa) in Fig. 6 were measured by a scanning densitometer, and expressed by arbitrary intensity units. Numbers in parentheses indicate ratios between the amounts of partial elongation products (88 kDa) formed with the heated and unheated DBPs. DBPs. The wild-type and ts DBPs were used either without any heat-treatment or after incubation a t 40 "C for 30 min. A constant amount (20 ng) of "H-labeled M13 DNA (344 cpm/ng) was mixed with increasing amounts of unheated or heated DBPs in 20 pI of T E M buffer containing 20 mM NaC1, 200 pg/ml RSA, and 10% glycerol. After overnight incubation a t 4 "C, the mixtures were filtered through nitrocellulose filters. Radioactivity retained on filters was determined by liquid scintillation counting and expressed as a percentage relative to the input counts. of individual DBP molecules to bind to ssDNA may be better examined using oligonucleotides; because only a limited number of DBP molecules can bind to an oligonucleotide, any cooperativity can be minimized. Since the oligonucleotidebinding capacity of the DBP has not been characterized, we first analyzed the DNA size requirement for binding. Synthetic oligonucleotides (8-84 nucleotides) were '"P-labeled and mixed with increasing amounts of wild-type DBP. The DBP -DNA complexes were separated from the free DNAs by agarose gel electrophoresis (Fig. 10). The patterns of the shift of DNA bands indicated that stable DBP.DNA complexes were formed with the 30-mer, 40-mer, and 84-mer DNAs. Complete shift of these oligonucleotides was achieved a t a DBP/DNA (w/w) ratio between 30 and 40, which is consistent with the expected saturation point assuming that one DBP molecule occupies 9-11 nucleotides (50). The DBP.24-mer complex appeared to be marginally stable. Smeared bands were observed with a 16-mer and excess amounts of DBP, indicating unstable binding. An octamer had no shift even a t a DBP/DNA ratio of 300. Binding to oligonucleotides under 24 bases could be demonstrated by UV cross-linking (data not shown).

Temperature-sensitive Adenovirus DNA-binding Proteins
Using the 84-mer as a DNA probe, we next examined the ts DBPs for their oligonucleotide-binding activity. The wildtype and mutant DBPs, unheated or heated to 40 "C for 30 Various amounts of wild-type DBP were mixed with 2 ng of '*Plabeled oligonucleotides (1-4 X lo4 cpm/ng) to yield the indicated DBP/DNA (w/w) ratios, and the DBP-DNA complexes were separated a t 4 "C by electrophoresis through a l% agarose gel. min, were mixed with "P-84-mer and electrophoresed at 4 "C ( Fig. 11). After incubation at 40 "C, the three ts DBPs formed no DBP-DNA complex band even at a DBP/DNA ratio of 40, indicating that all ts DBPs lost their oligonucleotide binding activity by heating. The wild-type DBP, in contrast, showed comparable binding activity before and after heating.

DBP/DNA , Unheated
Virtually identical results were obtained when a 30-mer was used instead of the 84-mer as a probe DNA (results not shown). We consistently observed that the three ts DBPs, when heated to 40 "C, formed one or two faint band(s) shifting between the free DNA and the DBP-DNA complex (Fig. 11). These bands are probably due to contamination with other ssDNA-binding proteins in the DBP preparations. Evidence for this came from several lines of experimentation. First, these bands were not stained with an anti-DBP monoclonal antibody (B6) in a Western blot analysis (Fig. 12). Second, when mixtures of the heated ts DBPs and 32P-84-mer were UV cross-linked and analyzed by SDS-PAGE, a major band of approximately 75-kDa was detected (data not shown). The unheated ts DBPs and 32P-84-mer, in contrast, formed a major band of about 100 kDa, which is close to the estimated molecular weight of the DBP.84-mer DNA complex. SDS-PAGE analysis showed that the DBPs themselves were not degraded after heating to 40 "C. Further, when the UV crosslinked samples were immunoprecipitated with B6 anti-DBP antibody, the 100-kDa band was quantitatively precipitated, but the 75-kDa band was not. All of these results indicate the presence of some contaminants with ssDNA-binding activity in the DBP preparations. This is not surprising because such contaminants cannot be completely removed by ssDNA-cellulose chromatography. Gel Mobility Shift Assay with M13 DNA-The results of the oligonucleotide-binding assay are seemingly contradictory to the findings obtained in the column binding and filterbinding assays. The discrepancy might be due to differences in either the assay methods or the DNAs used for each assay. To distinguish between these possibilities, we carried out another gel mobility shift assay with 32P-labeled M13 DNA. The results are shown in Fig. 13. Unlike oligonucleotides, the mobility of M13 DNA was progressively shifted as the number were mixed with 20 ng of 32P-labeled M13 DNA (4,800 cpm/ng) to give the indicated DBP/DNA ratios, and then electrophoresed at 4 'C through a 0.5% agarose gel.
of DBP molecules bound to the DNA increased. All three ts DBPs had significantly different patterns of shift before and after heating at 40 "C. However, the heated ts DBPs were still capable of shifting M13 DNA, and therefore appeared to retain some DNA-binding activity.

DISCUSSION
We examined three ts mutant adenovirus DBPs for their ability to support DNA replication and to bind to ssDNA. All three ts DBPs were unequivocally defective in adenovirus DNA replication at the nonpermissive temperature. The DBPs were further tested by several in vitro assays to determine whether the defect was in the initiation or elongation phase of DNA synthesis. In agreement with previous findings (16,26,30,36), our results in the partial elongation and synthetic template assays showed that the three ts DBPs had a severe defect in DNA chain elongation at the nonpermissive temperature. Furthermore, we were able to detect a defect in the initiation step, a finding that may have been overlooked previously (16,26,30) because the DBP is not an essential factor but only a weak stimulator for formation of the pTP dCMP initiation complex. Also, previous studies tested the ts DBPs at the permissive and nonpermissive temperatures. Since the rate of initiation complex formation differs depending on temperature, it is difficult to directly compare the activities of ts DBPs at the two temperatures. We found that the functional change of the mutant proteins at the nonpermissive temperature was irreversible. Taking advantage of this characteristic, we carried out the initiation assay at a constant temperature (30 "C) using the unheated and heated ts DBPs. The unheated ts DBPs and wild-type protein stimulated pTP dCMP synthesis %fold, whereas the heated ones were not stimulatory.
Results from various assays seemed inconsistent regarding the DNA-binding properties of DBP. The column binding assays using ssDNA-cellulose or ssDNA-agarose failed to show an unequivocal change in the relative DNA-binding affinity of the ts DBPs at the permissive and nonpermissive temperatures. The filter binding assay using M13 DNA

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showed that the three ts DBPs heated at 40 "C were still capable of retaining as much as 70% of the input DNA on the filters, although the amount of protein needed was significantly larger than that required by unheated proteins. Similar results were also obtained in the gel shift assay using M13 DNA. On the other hand, the gel shift assay using an 84-mer oligonucleotide clearly indicated that the ts DBPs lost their DNA-binding capacity by heating at 40 "C. These seemingly contradictory results may simply reflect the difference in the length of DNA used in each assay. Because of the cooperativity between DBP molecules (50,52), the overall binding affinity of the protein for an ssDNA molecule apparently increases as the number of DBPs bound to DNA increases. If one assumes that approximately 10 bases are covered by one DBP molecule (50), an 84-mer and M13 DNA could bind eight and 720 DBPs, respectively. Therefore, binding to oligonucleotides is far more sensitive to changes in the affinity of individual DBP molecules than is binding to large DNA molecules. Another explanation for the observed differences is that the ts DBPs may change the mode of DNA binding in such a way that a certain length of DNA is needed for binding. Electron microscopic observations that support such a possibility have recently been obtained.2 The seemingly conflicting results described above may actually explain the discrepancy between the conclusions of this and previous studies (35,36). Based on the change observed in oligonucleotide-binding activity, we suggest that the defect of ts DBPs in DNA replication results from their altered DNA-binding properties. Krevolin and Horwitz (35), using a filter-binding assay with denatured adenovirus DNA, showed that the ability of the Ad5ts107 DBP to bind to ssDNA did not significantly change between the permissive and nonpermissive temperatures and suggested that the inability of this mutant DBP to support DNA replication might reflect an alteration in DBP. Adpol interaction. The results of our filterbinding assay were similar to their results in that the ts DBPs had some DNA-binding activity at the nonpermissive temperature, even though we observed that the binding capacities of ts DBPs were significantly deteriorated after incubation at 40 "C.
The Ad2'NDlts23 DBP was originally described as having a drastically decreased DNA-binding affinity, yet was fully functional in DNA replication at the permissive temperature (30,36). In those studies, the mutant protein eluted from an ssDNA-cellulose column at 4 "C with a significantly lower concentration of NaCl(0.2 M) than that needed for wild-type DBP (0.6 M). We did not observe such a large difference despite repeating the column assay three times. However, we did observe that the NaCl concentration needed to elute Ad2'NDlts23 DBP varied over a considerable range (0.32-0.47 M NaCl), depending on the age of the column, although the relative difference with respect to the internal control was nearly constant in every run (0.10-0.14 M lower than 3H-wildtype DBP). Thus, the large difference reported in the previous studies appears to include the variation between separate column runs, in addition to the real difference between the wild-type and mutant DBPs. We believe that at the permissive temperature, the ability of Ad2'NDlts23 DBP to bind to ssDNA is strong enough to function normally in DNA replication. It is possible, however, that the column binding assay is measuring primarily electrostatic interactions between the DBP and ssDNA. The actual binding affinity is presumably composed of a combination of electrostatic force and hydrophobic interaction such as aromatic amino acid side chains stacking between the bases of DNA (55,56). Therefore, if the ~~~ M . Tsuji and G. R. Kitchingman, unpublished observations.

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Temperature-sensitive Adenovirus DNA-binding Proteins relative contribution of electrostatic forces to an overall DNAbinding affinity is greater for Ad2+NDlts23 than for wildtype DBP, it is possible that the mutant protein binds to ssDNA very poorly at high salt conditions, yet binds as tightly as wild-type DBP under the low salt condition in the DNA replication assay. In fact, the binding capacity of AdFNDlts23 DBP appeared to be indistinguishable from that of wild-type DBP under low salt conditions, as seen in the filter-binding and gel shift assays.
Except for the slight difference in the elution profile in ssDNA-cellulose chromatography, Ad2+NDlts23 and AdZtslllA DBPs were virtually identical in all characteristics studied. This would be expected because of the close proximity of the two mutations. Rather unexpectedly, the Ad5ts125 DBP also had very similar characteristics despite the fact that this mutation is located more than a hundred amino acids from the other two. However, Ad5ts125 DBP was inactivated at 40 "C more rapidly than the other two mutants. Moreover, the Ad5ts125 DBP produced at the nonpermissive temperature has been found to be degraded rapidly in vivo (24, 34), whereas the Ad2tslllA DBP appears to be stable (29,30). Thus, the mechanism of protein alteration by the Ad5ts125 mutation may somehow differ from that of the other two mutations. Several studies have indicated that the Ad5ts125 mutation affects not only the DNA replication function but also many other functions performed by DBP, such as transcriptional regulation (57), mRNA stability (58), virus assembly (59) and transformation (51). Whether the Ad2+NDlts23 and Ad2tslllA mutations also affect these other DBP functions is currently under investigation.
It is well established that the DBP consists of two functionally and structurally distinct domains. Mild chymotrypsin treatment separates the protein into 26-kDa amino-terminal and 44-kDa carboxyl-terminal fragments (47). The carboxylterminal fragment is still capable of binding to ssDNA (47) and is still fully functional in DNA replication (16). These findings are consistent with the fact that the three ts DBPs have mutations within their carboxyl-terminal domain. Although DBPs from Ad2 and Ad5 differ by nine amino acids (31), these differences are in the amino-terminal domain; the carboxyl-terminal domains are identical. The DBPs of these two serotypes can be used interchangeably in the in vitro DNA replication assays using components prepared from reciprocal Ad serotypes.
How does the altered DNA binding at the nonpermissive temperature give rise to the defect in DNA replication? As mentioned above, all three ts DBPs appear to be defective in both the initiation and elongation phases of DNA replication. The role of the DBP in the initiation of replication is complex. Although not essential for the initiation process, the DBP stimulates the formation of pTP. dCMP initiation complex in the presence of NFI. Part of the reason for this has been explained by recent studies (3, 53) showing that the DBP enhances the affinity of NFI for its binding site at the origin of replication. This enhanced NFI binding appeared to be an indirect effect accompanied by the binding of DBP to DNA (3,53), although the possibility of direct DBP-NFI interaction cannot be excluded. Cooperation with NFI, however, is not the only role of DBP in initiation, since many studies have shown the stimulation of initiation by DBP in a system consisting of purified components free from NFI (3,18,54). Thus, the DBP may have other effects, such as enhancement of the binding of Adpol. pTP to the end of adenovirus DNA and assistance in unwinding the terminal regions of the template DNA prior to initiation.
In contrast to its unknown role in initiation, the function of the DBP in DNA chain elongation has been relatively well defined. The DBP acts as a facilitator of Adpol and makes the enzyme highly processive (45). Stimulation of Adpol by the DBP can result from either a direct interaction between the two proteins or an indirect effect, wherein the DBP changes the topology of template DNA to one more favorable for the polymerase. Although there is circumstantial evidence for a direct Adpol .DBP interaction (45), attempts to isolate the Adpol. DBP complex have not been successful. In the elongation process of DNA synthesis, and perhaps also in the initiation process, the DBP has to bind to single-stranded regions of template DNA, although it is obvious that the binding to DNA per se is not sufficient to promote DNA synthesis. Other, functionally equivalent DBPs such as E. coli single-stranded DNA-binding protein, cannot substitute for the Ad DBP (17,18,45). Based on the results of the DNA-binding assays, we propose that at the nonpermissive temperature, the three ts DBPs either no longer bind to the template DNA, or bind to the template DNA in an aberrant fashion. The alternatives are experimentally distinguishable; in the former case, the defective ts DBPs should be readily complemented by wild-type DBP; in the latter, the binding of altered ts DBPs will generate an aberrant template DNA which may be inactive even in the presence of wild-type DBP (assuming that the binding of altered ts DBPs is tight enough to inhibit the binding of wild-type DBP). We have obtained preliminary results supporting the latter possibility using the synthetic template assay, which showed that DNA synthesis with wildtype DBP was inhibited by the addition of the Ad5ts125 DBP preincubated at 40 "C for 30 min in a dose-dependent fashion. However, no such inhibition was observed in the in vitro replication assay using the authentic adenovirus DNA-protein complex (Fig. 5). Therefore, further work in this area is clearly required. In addition, it should be emphasized that this explanation does not necessarily imply that the defect in ts DBPs is confined to DNA-binding properties. Whether the ts DBPs have aberrations in possible interactions with Adpol and NFI remains an open question.
In summary, we have shown that the inability of the three ts mutant DBPs to support DNA replication at the nonpermissive temperature can be ascribed to their altered DNAbinding properties. Further work will be required to determine the nature of the changes to the DBP that result in these altered binding properties.