THE JOURNAL OF BKXOGICAL. CHEMISTRY Vol. 265, No. 13, Issue of May 5. pp. 7693-7700.1990 0 1990 by The American Society for Biochemistry and Molecular Biology, Inc. Printed &R U.S.A.

The Role of Human Single-stranded DNA Binding Protein and Its Individual Subunits in Simian Virus 40 DNA Replication*

(Received for publication, November 14, 1989)

Mark K. Kenny, Uwe SchlegelS, Henry Furneauxz, and Jerard Hurwitz From the Graduate Program in Molecular Biology, and the $Cotzias Laboratory of Neuro-oncology, Sloan-Kettering Cancer Center, New York, New York 10021

Human single-stranded DNA binding protein (hu- man SSB) is a multisubunit protein containing poly- peptides of 70, 34, and 11 kDa that is required for SV40 DNA replication in vitro. In this report we iden- tify the functions of the SSB and its individual subunits in SV40 DNA replication. The 70 kDa subunit was found to bind to single-stranded ‘DNA, whereas the other subunits did not. Four monoclonal antibodies against human SSB were isolated which inhibited SV40 DNA replication in vitro. The antibodies have been designated aSSB70A, aSSB70B, aSSB70C, and aSSB34A to indicate which subunits are recognized. Immunolocalization experiments indicated that human SSB is a nuclear protein. Human SSB is required for the SV40 large tumor antigen-catalyzed unwinding of SV40 DNA and stimulates DNA polymerases (pol) a and 6. The DNA unwinding reaction and stimulation of ~016 were blocked by aSSB70C, whereas the stimula- tion of pola! by human SSB was unaffected by this antibody. Conversely, aSSB70A, -7OB, and -34A in- hibited the stimulation of pola, but they had no effect on DNA unwinding and ~016 stimulation. None of the antibodies inhibited the binding of SSB to single- stranded DNA. These results suggest that DNA un- winding and stimulation of pola and ~016 are required functions of human SSB in SV40 DNA replication. The human SSB ‘IO-kDa subunit appears to be required for DNA unwinding and ~016 stimulation, whereas both the 70- and 34-kDa subunits may be involved in the stimulation of pola.

The in vitro SV40 DNA replication system has provided an assay for the identification and isolation of a single-stranded DNA binding protein from human cells (human SSB)l which is analogous to the SSBs from Escherichia coli and bacterio- phage T4 (T4 gene 32 protein) (Wobbe et al., 1987; Wold and Kelly, 1988; Fairman and Stillman, 1988). The role of human SSB in SV40 DNA replication, and perhaps by analogy, cellular DNA replication, is now being investigated.

SV40 depends almost entirely on the host cell to provide its replication machinery (for recent reviews, see Stillman,

* This work was supported by Grant GM34559 from the National Institutes 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 “aduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 The abbreviations used are: SSB, single-stranded DNA binding protein; SV40, simian virus 40; TAg, SV40 large tumor antigen; pola, DNA polymerase ol; polb, DNA polymerase 6; PCNA, proliferating cell nuclear antigen; BSA, bovine serum albumin; T4g32, bacterio- phage T4 gene 32 protein; IgG, immunoglobulin G; BrdUrd, bromo- deoxyuridine.

1989; Challberg and Kelly, 1989; Hay and Russell, 1989). However, the sole viral protein required for replication, SV40 large tumor antigen (TAg), provides multiple functions in- cluding origin binding and melting and DNA helicase activi- ties (Tjian, 1978; Deb and Tegtmeyer, 1987; Borowiec and Hurwitz, 1988a, 198813; Stahl et al., 1986). In the presence of ATP at 37 “C, TAg forms a bilobed, nucleoprotein structure at the SV40 origin of replication in which the origin DNA is distorted and locally melted (Dean et al., 1987c; Mastrangelo et al., 1989; Borowiec and Hurwitz, 1988b; Roberts, 1989; Dean et al., 1989). The addition of human SSB and topoisom- erase I or II leads to extensive unwinding of the DNA (Dean et al., 1987a, 1987b; Dodson et al., 1987; Wold et al., 1987), although this unwinding may normally be coupled to DNA synthesis. Subsequent events in SV40 DNA replication are unclear, but several proteins have been implied to play specific roles. DNA polymerase a-primase complex (pola-primase) is likely to be involved in the initiation reaction and lagging strand synthesis, whereas DNA polymerase 6 (pals), prolifer- ating cell nuclear antigen (PCNA), and other auxilliary fac- tors have been implicated in leading strand synthesis (Murak- ami et al., 1986, Prelich et al., 1987; Prelich and Stillman, 1988; Downey et al., 1988; Lee et al., 1989a, 1989b; Wold et al., 1989; Tsurimoto and Stillman, 1989). RNA primers can be removed by the combined action of RNase H and 5’ + 3’ exonuclease. After gap-filling by one of the polymerases, DNA ligase can seal nicks, and topoisomerase II can decantenate daughter molecules (Ishimi et al., 1988; Yang et al., 1987).

In addition to the role of human SSB in DNA unwinding, which presumably reflects the ability to bind to and stabilize single-stranded DNA, human SSB can stimulate the activity of pola and ~016 (this report and Kenny et al., 1989). In this report, the role of human SSB and its individual subunits in these reactions and in SV40 DNA replication has been inves- tigated using anti-SSB monoclonal antibodies and other tech- niques.

MATERIALS AND METHODS

Reagents-The following reagents were obtained commercially: p~ly(dA)~~~ (Life Sciences); micrococcal nuclease, E. coli SSB, T4g32, Sephadex G-25, and ohgo(dT), (Pharmacia LKB Biotechnology Inc.); radionucleotides (Du Pont-New England Nuclear); unlabeled nucleotides, M13mp18 DNA, Ml3 sequencing primer (dTCCCAG- TCACGACGT), DNase I, and E. coli DNA polymerase I (Boehringer Mannheim); creatine phosphokinase (Worthington); chloroquine (Sterling Drug Inc.); protein A-Sepharose (Sigma); goat anti-mouse agarose (HyClone); immunoblotting reagents (Bio-Rad); immunohis- tochemical reagents (Vector Laboratories). Bovine serum albumin (BSA, Miles Laboratories) was heat-denatured before use. TAE buffer (1 X) is 40 mM Tris acetate and 1 mM EDTA.

DNA and Replication Proteins-The SV40 origin-containing plas- mid pSVOlAEP has been described previously (Wobbe et al., 1985). Crude extract (Wobbe et aZ., 1985), topo I (Ishimi et al., 1988), pol~l- primase (Ishimi et al., 1988), PCNA-dependent ~016 (Lee et al., 1989b),

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7694 Role of Human SSB in SV40 DNA Replication

PCNA (Lee et a!., 1988), and the 0.4 M double-stranded DNA cellulose inhibitor/activator I fraction (Lee et a&, 1989a) were prepared from HeLa cells as described elsewhere. TAg was immunoaffinity-purified (Mastrangelo et al., 1989) from insect cells infected with a TAg- encoding, recombinant baculovirus vector (O’Reilly and Miller, 1988). Adenovirus DNA binding protein was purified from adenovirus- infected HeLa cells (Ikeda et al., 1981).

Human SSB was isolated from 1.2 x 10” HeLa cells by a modifi- cation of the procedure of Wobbe et al. (1987). One unit of human SSB supported the incorporation of 1 nmol of dTMP in 60 min in the SV40 DNA replication assay (Wobbe et al., 1987). HeLa crude extract (9.6 g of protein, 6600 units, 600 ml) was fractionated by O- 35% ammonium sulfate precipitation as described previously (Wobbe et al., 1987). The AS35 fraction (2.9 g of protein, 5900 units, 144 ml) was adjusted to 0.5 M NaCl in buffer BN (20 mM Tris-HCl, pH 7.5, 10% glycerol, 0.1 mM EDTA, 0.01% Nonidet P-40, 1 mM dithiothre- itol) and filtered through filter paper (Whatman 2). This fraction was loaded onto a 6-ml (-0.5 g of protein/ml bed volume) single-stranded DNA cellulose column (3 mg of DNA/g of cellulose, Sigma) equili- brated with buffer BN containing 0.5 M NaCl. A short, squat column (2.5 X 10 cm, Kontes) was used to prevent clogging such a small amount of resin with the crude fraction. The column was washed extensively (-10 bed volumes) with buffer BN containing 0.5 M NaCl. Active fractions were eluted with buffer BN containing 2 M NaCl. The SSB (3.7 mg protein, 2400 units, 5.8 ml), which was >80% pure at this stage, was diluted with buffer BN to 50.1 mg protein/ml and 550 mM NaCl and applied to a 2-ml phosphocellulose column equil- ibrated with buffer BN containing 50 mM NaCl. After washing with 10 ml of buffer BN containing 50 mM NaCl, activity was eluted with buffer BN containing 0.25 M NaCl. Active fractions were pooled and dialyzed against buffer BN (20 mM HEPES-NaOH, pH 7.0, 25% glycerol, 0.1 mM EDTA, 0.01% Nonidet P-40, 1 mM dithiothreitol) containing 0.25 M NaCl and stored at -80 “C (1.8 mg of protein, 1300 units, 1.1 ml). Recently, a fast protein liquid chromatography Mono Q column was substituted for the phosphocellulose column in order to remove contamination by a small amount of nuclease activity which inhibited replication reactions containing purified proteins. The Mono Q column was loaded and washed with 0.1 M NaCl in buffer BN and was eluted with a linear gradient from 0.1 to 0.5 M NaCl in the same buffer. The SSB eluted at 0.26 M NaCl.

The key feature of this purification procedure was the loading of the single-stranded DNA cellulose column in high salt which resulted in >99.8% of the protein flowing through the column and >300-fold purification. Previously, a double-stranded DNA cellulose column has been used (Wobbe et al., 1987), hut it was found to have a limited SSB binding capacity, suggesting that the SSB may have bound to regions of single-stranded DNA on the resin. SSB-containing frac- tions could be pooled based on protein determinations rather than activity profiles in order to hasten the procedure. Concentrated preparations of human SSB were found to precipitate out of solution in low salt and thus were stored in 0.25 M NaCl. This purification procedure was rapid and reproducible and resulted in relatively pure, concentrated, and soluble SSB.

Labeled DNAs-“P-Labeled DNA was prepared by primer exten- sion using Ml3 as a template. Reaction mixtures (50 ~1) contained 50 mM Tris-HCl, pH 7.5, 10 mM MgSO,, 1 mM dithiothreitol, 0.5 mg/ ml BSA, 0.5 pg of M13mp18 DNA, 1.5 ng of sequencing primer (15 mer), 40 pM each of dATP, dGTP, dTTP, and [a-32P]dCTP (300 PCi), and 5 units of E. coli DNA polymerase I. When the DNA was used for UV cross-linking experiments, bromo-dUTP was substituted for dTTP. Reactions were incubated for 30 min at 30 “C followed by isolation of the DNA by phenol/chloroform extraction and Sephadex G-25 spin gel filtration. The DNA was heat-denatured at 100 “C for 5 min and had a specific activity of 50,000 cpm/ng.

CJV Cross-linking-UV cross-linking reactions (50 ~1) containing 50 mM Tris-HCl, pH 7.5, 5 mM MgCl,, 0.5 mM dithiothreitol, 0.2 mg/ ml BSA, 4 ng of single-stranded 32P-labeled BrdUrd-DNA, and 100 ng of the indicated SSB were incubated at 37 “C for 10 min. Reaction mixtures were spotted on Saran Wrap on top of a Photoproducts TM-36 mid-range UV transilluminator (302 nm; 8000 pW/cm*) and illuminated for 4 min at room temperature. Mixtures were then transferred to 1.5-ml Eppendorf test tubes, and 1.0 pg of DNase I, 5 units of micrococcal nuclease and CaClz to 1.5 mM were added. Reactions were incubated at 37 “C for 20 min and stopped by addition of 50 ~1 of a solution containing 180 mM sodium pyrophosphate and 50 mM EDTA. Proteins were precipitated with 10% trichloroacetic acid, washed with ether, and resuspended in SDS-PAGE sample buffer.

DNA Binding Assay-Nitrocellulose filters were pretreated with alkali to reduce nonspecific binding of DNA which was not protein- mediated (McEntee et al., 1980). DNA binding reactions (50 pl), containing 50 mM Tris-HCl, pH 7.5, 5 mM MgCl*, 0.5 mM dithiothre- itol, 0.2 mg/ml BSA, 4 ng of single-stranded [32P]DNA, and HeLa SSB or isolated subunits, were incubated for 10 min at 37 “C. Mix- tures were filtered through nitrocellulose filters and washed with 1 ml of 50 mM Tris-HCl, pH 7.5, 5 mM MgCl,. Radioactivity retained on the filters was determined by liquid scintillation spectroscopy.

Mono&ma1 Antibodies-Mouse hybridomas were developed com- mercially by American Biotechnologies Inc. using purified human SSB as the antigen. Hybridomas were grown in serum-free media, and the supernatant was collected. The isotype of all four antibodies characterized here was found to be IgGlk, although some in uitro class switching was observed. Hybridoma supernatant was adjusted to 50 mM Tris-HCl, pH 8.5, 3 M NaCl and loaded onto a protein A- Sepharose column. The column was washed with 10 mM Tris-HCl, pH 8.5, 3 M NaCl, and antibodies were eluted with 100 mM glycine HCl, pH 2.5. Purified antibodies were neutralized and dialyzed against phosphate-buffered saline and stored in small aliquots at -80 “C.

Zmmunohistochemical Analysis-Cultured human cells (HEp-2) fixed on slides (Behring Diagnostics Inc.) were incubated with 2 pg/ ml purified monoclonal antibody for 18 h at 4 “C. After washing with phosphate-buffered saline, antibody binding was detected by the sequential application of biotinylated horse anti-mouse secondary antibody and avidin-biotin peroxidase complex (Hsu et al., 1981). Peroxidase was visualized by incubation with diaminobenzidine and photographed using phase contrast microscopy.

RESULTS

The Structure of Human SSB-Human SSB (also referred to as HeLa SSB, RF-A, and RP-A) has been purified from HeLa and 293 cells based on its ability to support SV40 DNA replication in an in uitro eomplementation assay (Wobbe et al., 1987; Fairman and Stillman, 1988; Wold and Kelly, 1988). Based on the recovery of SSB replication activity from crude extracts, a minimum of 50,000 SSB molecules/HeLa cell is estimated. This number may be an underestimate if only a fraction of the SSB was extracted from the cell. As shown in Fig. 1 and previously (Fairman and Stillman, 1988; Wold and Kelly, 1988), SSB purified to apparent homogeneity from human cells consists of 3 tightly associated polypeptides of approximately 70, 34, and 11 kDa. The largest subunit mi- grated as a smear or two to three distinct bands depending on the resolution of the SDS-PAGE and regardless of the pres- ence of reducing agents (Fig. 1; Wobbe et al., 1987; and data not shown). The “70-kDa bands” are immunologically related to each other and may represent post-translational modifica- tions. In contrast, the 70-, 34-, and ll-kDa subunits are antigenically distinct (data not shown). Human SSB has an isoelectric point of 5.6.’ Gel filtration chromatography and glycerol gradient centrifugation of human S$B indicated that the protein has a Stokes radius of about 52 A and a sedimen- tation coefficient of approximately 5.1 S (Wobbe et al., 1987; Fairman and Stillman, 1988; Wold and Kelly, 1988; and data not shown). Assuming a partial specific volume of 0.725 cm3/ g, a native molecular mass of 110 kDa and a frictional ratio of 1.65 can be calculated by the method of Siegel and Monty (1966). These data are consistent with SSB having a some- what prolate structure and a subunit stoichiometry of 1:l:l. Further studies will be required to better define the structure of human SSB. Human SSB was observed to aggregate at a low ionic strength and high SSB concentrations. Other SSBs also have the tendency to aggregate, and this property may be related to the cooperative manner in which SSBs usually bind to DNA (Chase and Williams, 1986; Williams and Chase, 1989). The mechanism by which human SSB binds to DNA is not well characterized.

2 G. Hodgins and J. Hurwitz, unpublished observations.

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Role of Human SSB in SV40 DNA Replication

45 -

- -34 31 -

top- . 200-

93 - rm Ad DBP,

=HSSB-70 66 -

45 - .o - T4g32

31 - - HSSB-34

22 - e -E. SSB

14 - - HSSB-11

22 - front -

1234

14-w -11

FIG. 1. SDS-polyacrylamide gel electrophoresis of human SSB. Electrophoresis of 0.5 rg of human SSB was carried out according to the method of Laemmli (1970) using a 4% stacking gel and a 14% separating gel. The 70-, 34-, and 11-kDa subunits are labeled on the right. Molecular mass markers are indicated on the left: phosphorylase b (93 kDa), bovine serum albumin (66 kDa), ovalbumin (45 kDa), carbonic anhydrase (31 kDa), soybean trypsin inhibitor (22 kDa), lysozyme (14 kDa). The gel was silver-stained using a kit from ICN Biochemicals.

The Human SSB 70-kDa Subunit Binds to Single-stranded DNA-Human SSB binds tightly to single-stranded DNA (Wobbe et al., 1987; Fairman and Stillman, 1988; Wold and Kelly, 1988; Wold et al., 1989). In order to determine which of the subunits binds to DNA, UV photocross-linking exper- iments were performed with human SSB. Other SSBs (ade- novirus DNA binding protein, E. coli SSB, and T4g32) were also tested as controls. Individual SSBs were incubated with 32P-labeled, BrdUrd-substituted, single-stranded DNA. Fol- lowing UV cross-linking, SSB. DNA complexes were treated with nucleases to digest all but a short DNA fragment pro- tected by the SSBs. Thus, the SSB or SSB subunit bound to the DNA was labeled by this technique. The SSB.oligonucle- otide complexes were then electrophoresed on SDS-polyacryl- amide gels followed by autoradiography (Fig. 2). Only the 70- kDa subunit of human SSB was labeled by this method, indicating that it was bound to the DNA, whereas the 34- and ll-kDa subunits were not in direct contact with the DNA. The adenovirus DNA binding protein (adenovirus DNA bind- ing protein, 72 kDa) and the T4 gene 32 protein (T4g32, 33.5 kDa) yielded labeled bands of the appropriate sizes. However, the rate of migration of the proteins was somewhat reduced by the attached DNA. The 19-kDa E. coli SSB, which binds to DNA as a tetramer, produced a more complex series of bands corresponding to one or more protomers of the SSB.

Although the results presented in Fig. 2 indicate that the human SSB 70-kDa subunit binds to DNA, it is possible that the other subunits may be required for this activity. To test this possibility, the individual human SSB subunits were isolated by SDS-PAGE and were renatured separately or together by the procedure of Hager and Burgess (1980). The subunits were then tested for their ability to bind single- stranded DNA using a nitrocellulose filter binding assay (Fig. 3). The 70-kDa subunit exhibited DNA binding activity in

top -

200 -

93 -

Y

66 - 1 Ad DBP,

HSSB-70

45 -

0 - T4g32

front - 0

1234

FIG. 2. UV cross-linking of SSBs to “P, BrdUrd-DNA. The indicated SSBs were incubated with ‘“P-labeled, BrdUrd-substituted single-stranded DNA and exposed to UV light as described under “Materials and Methods.” After nuclease treatment, the SSBs were electrophoresed on SDS-polyacrylamide gels followed by autoradiog- raphy. A, 16% SDS-PAGE. B, 10% SDS-PAGE. Molecular mass markers are indicated on the left in kilodaltons. The positions of unlabeled SSBs are indicated on the right. Note that SSBs cross- linked to short DNA fragments migrate slightly slower than free SSBs. HSSB-70, -34, and -II denote the human SSB 70-, 34., and 11-kDa subunits, respectively. Ad DBP, adenovirus DNA binding proteins.

50 -

11 ho0

0 I fg34kOO 0 1 2 3 4

HeLa SSB Fraction(~ll

FIG. 3. DNA binding activity of the isolated human SSB subunits. The 70-, 34-, and 11-kDa subunits of the human SSB were separated by SDS-PAGE, extracted from gel slices, and renatured separately or in combination by the procedure of Hager and Burgess (1980). The SSB subunits were tested for their ability to bind single- stranded IR’P]DNA in a nitrocellulose filter binding assay as described under “Materials and Methods.” The concentration of the isolated subunits could not be directly determined because of the presence of bovine serum albumin in the buffers. However, based on SDS-poly- acrylamide gel analysis, there was approximately equal recovery of each of the subunits. Thus, equal volumes of the isolated human SSB subunits contained the same ratio of polypeptides as found in the intact SSB protein. The SSB subunits assayed were as indicated: 0, 70-, 34-, and ll-kDa subunits; 0, ‘IO-kDa subunit; n , 34.kDa subunit; 0, 11-kDa subunit.

the presence or absence of the other subunits. The 34- and 11-kDa subunits showed little or no DNA binding activity, and they did not significantly alter the ability of the 70-kDa subunit to bind DNA. We have thus far been unable to

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7696 Role of Human SSB in SV40 DNA Replication

reconstitute SV40 DNA replication activity with the isolated human SSB subunits.

Monoclonal Antibodies against Human SSB-Mice were immunized with purified SSB from HeLa cells, containing the three subunits of 70, 34, and 11 kDa. Hybridomas were isolated which produced monoclonal antibodies which reacted with human SSB in an enzyme-linked immunoassay. The four antibodies discussed in this paper were further charac- terized by immunoblotting. Three of the antibodies recognized the 70-kDa subunit, whereas one reacted with the 34-kDa subunit (Fig. 4). They have been designated &SB’IOA, -7OB, 7OC, and -34A, where N signifies “anti” and the number denotes the reactive SSB subunit. Although all four antibodies worked to some extent in the immunoblotting assay, &SB70A, -7OB, and -70C generally gave a weak signal sug- gesting that they may recognize epitopes in the native protein which are lost upon denaturation. In contrast, &SB34A always gave a strong signal in the immunoblotting assay.

Human SSB Is a Nuclear Protein-The monoclonal anti- bodies were used to determine the intracellular localization of SSB. After fixation, cells were treated with (YSSB antibodies and visualized by immunoperoxidase staining and phase con- trast microscopy (Fig. 5A-C). SSB was found almost exclu- sively in the nucleus and produced a distinctive punctate staining pattern. Examination of other cell lines or tissue k L sections or the use of bright field microscopy also indicated that the SSB was located in the nucleus. Substitution of the c&SB antibodies with nonimmune mouse IgG produced no staining (Fig. 50).

(uSSB Monoclonal Antibodies Specifically Inhibit S V40 DNA Replication in Vitro-Purified &SB monoclonal antibodies were tested for their ability to inhibit SV40 DNA replication using crude extracts of HeLa cells (Fig. 6). All four antibodies inhibited replication to varying extents. Relatively low levels of (YSSB~OA and -7OB antibodies inhibited replication, but a maximum of 75% inhibition was observed. The effect of cuSSB34 plateaued at 85% inhibition, while aSSB70C de- creased nucleotide incorporated by more than 95% at high

FIG. 5. Cellular localization of human SSB. HEp-2 cells were incubated with &SB’IOA (A), nSSB70C (B), &SB34A (C), or non- immune mouse IgG (D) and visualized by peroxidase staining and phase contrast microscopy. Note that some cells have apparently fused to form syncytia and thus are multinucleated.

1234

70- -

34-

11 -

I

I

FIG. 4. Identification of human SSB subunits recognized by aSSB monoclonal antibodies. Purified human SSB was subjected to SDS-PAGE on a 14% gel and then transferred to nitrocellulose by electroblotting. Nitrocellulose strips were incubated sequentially with &SB hybridoma supernatant, alkaline phosphatase-conjugated goat anti-mouse antibody, and alkaline phosphatase color development reagents. Lane 1, &SB70A; lane 2, uSSB70B; lane 3, aSSB70C; lane 4, olSSB34A.

Ab Added

FIG. 6. Inhibition of SV40 DNA replication by aSSB mono- clonal antibodies (Ab). HeLa crude extract (170 pg of protein) and purified monoclonal antibodies were incubated at 0 “C for 30 min in a volume of 13 ~1. The remaining reaction components were added such that final reaction mixtures (30 ~1) contained 40 mM creatine phosphate-diTris salt, pH 7.7, 7 mM MgCl,, 0.5 mM dithiothreitol, 4 mM ATP, 200 pM each of UTP, GTP, and CTP, 100 WM each of dATP, dGTP, and dCTP, 20 pM [“H]dTTP (300 cpm/pmol), 1 pg of creatine phosphokinase, 180 ng of pSVOlAEP DNA, 10 pg of BSA, and 1.0 tig of TAR. Following incubation at 37 “C for 60 min. acid- insoluble radioac&ity was determined. 0, olSSB70A; 0, (uSSB34A; W, nSSB70B; 0, &SB70C; A, equimolar combination of c&SB34A, -7OB, and -70C (totaling the indicated value).

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Role of Human SSB in SV40 DNA Replication 7697

levels of antibody. A combination of three of the antibodies (34A, 70B, and 7OC) completely abolished DNA synthesis.

Replication reactions inhibited (70-100%) of the aSSB antibodies were at least partially reconstituted by the addition of purified human SSB (Fig. 7). Although the addition of SSB did not completely reverse the effect of &SB70B and -34A, human SSB did stimulate reactions inhibited by these anti- bodies 4- to 8-fold. It is possible that addition of purified SSB might not fully restore replication activity if the SSB-anti- body complex is not only inactive, but also inhibitory to SV40 DNA replication. These results indicate that the inhibition of SV40 DNA replication by the cvSSB antibodies was caused specifically by the neutralization of human SSB in the crude extract.3

The Antibodies Do Not Inhibit DNA Binding-The effect of aSSB monoclonal antibodies on the binding of human SSB to 32P-labeled, single-stranded DNA was tested using a nitro- cellulose filter binding assay. In the absence of SSB the DNA passed through the filter, whereas in the presence of limiting levels of human SSB approximately 25% of the DNA was retained (Table I). None of the antibodies inhibited the bind- ing of SSB to DNA, and the antibodies alone, in the absence of SSB, did not exhibit any DNA binding activity. Higher levels of antibodies (up to 3 pg of antibody:1 ng of SSB) did not affect DNA binding (data not shown). Although the 70- kDa SSB subunit binds to DNA, &SB70A, -7OB, and -70C antibodies must react with epitopes which are not required for DNA binding.

Since the aSSB monoclonal antibodies specifically recog- nize SSB but do not inhibit DNA binding, it is possible that an antibody. SSB . DNA complex can form. The formation of a tertiary complex between &SB antibodies, SSB, and 32P- labeled, single-stranded DNA was tested by immunoprecipi- tation with goat anti-mouse IgG (Table II). Levels of nonspe- cific binding (i.e., in the absence of SSB or antibody) were

3 In the experiments described in Figs. 6 and 7, crude extracts were first preincubated with the antibodies at 0 “C followed by the addition of reagents essential for the replication reaction. The addition of large amounts of human SSB partially reversed the effects of anti- bodies 70B and 34A (Fig. 7). At the level of antibody used (0.45 pg), the addition of human SSB prior to the addition of crude extracts did not alter the results described in Fig. 7. High levels of SSB only partially reversed the effects of antibodies 70B and 34A. These results could be due to the presence of nonspecific inhibitors in these two antibody preparations. We consider this unlikely for the following reasons: reduction of these two antibody preparations (the use of 0.2 rg) did not alter the extent of inhibition of replication (90 and 80% with 34A and 70B, respectively). At this concentration, preincubation of the antibodies with increasing concentrations of human SSB reversed the inhibition. Thus when 0.2 pg of antibody 34A was preincubated for 15 min at 0 “C, with 0, 0.7, and 1.5 rg of human SSB, followed by incubation with crude extracts for 30 min at 0 “C, replication was inhibited 82, 40, and 23%, respectively; when the antibody was preincubated with 3 pg of human SSB, the replication reaction was stimulated 32%. As noted in Fig. 7, we have consistently observed the replication reactions with crude extracts can be stimu- lated by supplementation with human SSB. Similar results were obtained with the antibody 70B. High levels of antibodies (1 pg and higher in some cases) did not affect a number of individual enzymatic reactions examined. Thus the human SSB-dependent DNA unwind- ing reaction catalyzed by T antigen and the SSB-stimulated elonga- tion of primed DNA templates by DNA polymerases 01 and 6, when affected, required high concentrations of antibodies. This suggests that the SSB-antibody complexes were capable of participating in these individual reactions. If each reaction (unwinding, elongation, etc.) were partially inhibited, the product of these individual effects could be greater than the sum of each effect. Since all of these reactions are essential for SV40 DNA replication, this would explain the more pronounced inhibitory effect of low levels of these antibodies on the overall replication reaction than their effect on individual reactions.

. 706

P I I I I , I 0 1 2 3 4 5 6

Human SSB Added trg)

FIG. 7. Reconstitution of inhibited replication reactions by addition of purified human SSB. HeLa crude extract (170 pg of protein) and purified monoclonal antibodies (Ab) (0.45 pg) were incubated with various amounts of human SSB for 30 min at 0 “C in a volume of 15.5 pl. The remaining reaction components were added as in Fig. 6, and reaction mixtures were incubated for 60 min at 37 “C. V, no antibody added; 0, crSSB70A; 0, olSSB34A; n , otSSB70B; Cl, olSSB70C; A, combination (comb) of olSSB34A, -7OB, and -70C (0.15 fig of each).

TABLE I

Effect of LYSSB monoclonal antibodies on the binding of human SSB to single-stranded DNA

The effect of antibodies on the binding of SSB to single-stranded [32P]DNA was tested using a nitrocellulose filter binding assay. Re- action mixtures (49 ~1) contained 50 mM Tris-HCl, pH 7.5, 50 mM NaCl, 5 mM MgCl,, 0.5 mM dithiothreitol, 10 pg of BSA, and 1.0 ng of human SSB and 30 ng of purified monoclonal antibody as indicated. Reactions were incubated at 0 “C for 30 min followed by addition of (1 ~1) 12 pmol (as nucleotide) of 32P-labeled, single-stranded DNA and further incubation at 37 “C! for 10 min. Reaction mixtures were filtered on nitrocellulose filters and washed with 1 ml of a solution containing 50 mM Tris-HCl, pH 7.5, 50 mM NaCl, and 5 mM MgClz and radioactivity retained on the filters was determined. The amount of SSB used in these experiments was in the linear assav range.

Human SSB added

Antibody added DNA retained

- -

70A 34A 70B 7oc

70A.34A.70B.m 70C

pm01 co.3

2.8 3.0 2.9 3.2 3.2

co.3

significant but relatively constant. A tertiary complex was clearly formed when &SB70A, -34A, or -7OB was used. Sur- prisingly, no complex was detected between &SB70C, SSB, and DNA. Increasing the level of &SB70C or altering the order of addition of antibody, SSB, and DNA did not change this result. Similar results were obtained using a gel mobility shift assay.“ A slow-migrating complex between SSB and single-stranded DNA was detected, and the migration of this complex was further retarded by aSSB70A, -34A, and -7OB. However, aSSB70C had no detectable effect on the migration of the SSB . DNA complex. This suggests that this antibody neither inhibited DNA binding nor did it stably bind to the SSB . DNA complex.

aSSB70C Inhibits SV40 DNA Unwinding-The effect of c&SB antibodies on unwinding was tested in reactions con- taining TAg, human SSB, topoisomerase I, and SV40 origin- containing DNA (Fig. 8). DNA unwinding was virtually abol- ished by &SB70C, whereas the other antibodies had little or no effect. The ratio of otSSB70C antibody to SSB required to

4 L. Clark and J. Hurwitz, unpublished observations.

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7698 Role of Human SSB in SV40 DNA Replication

TABLE II

Immunoprecipitation of nSSB antibody. SSB. DNA complexes Goat anti-mouse IgG agarose was used to detect the formation of

a tertiary complex between &SB monoclonal antibodies, human SSB, and single-stranded DNA. Reaction mixtures (50 ~1) contained 50 mM Tris-HCl, pH 7.5, 50 mM NaCl, 5 mM MgC12, 0.5 mM dithiothreitol, 10 fig of BSA, 12 pmol (as nucleotide) of “P-labeled, single-stranded DNA, and as indicated 5.0 ng of human SSB and 30 ng of purified &SB monoclonal antibody. Reactions were incubated at 0 “C for 20 min and then at 37 “C for 10 min. After addition of 100 ~1 of goat anti-mouse IgG agarose (50 gl packed volume), reaction mixtures were incubated at 25 “C for 30 min with occasional mixing. Suspensions were centrifuged, and pellets were washed twice with 0.5 ml of a solution containing 50 mM Tris-HCl, pH 7.5, 50 mM NaCl, and 5 mM MgCl,. The amount of DNA retained in the pellet was determined by measuring Cerenkov radiation.

&SB antibody added

Human SSB added DNA precipitated

70A 34A 70B 7oc

- 70A 34A 70B 7oc

pm01 1.8 2.0 2.0 2.1 1.5 5.0 5.3 7.2 1.8

inhibit unwinding activity was similar to the ratio needed to suppress the replication reaction.

aSSB70A, -7OB, and -34A Inhibit the Stimulation of Pola by Human SSB-Human SSB stimulates DNA synthesis by polcv approximately lo-fold using poly(dA) oligo(dT) as a template under the same conditions used for DNA replication and unwinding reactions. The monoclonal antibodies aSSB70A, -7OB, and -34A blocked the stimulation of polcv by SSB by 50-65% (Fig. 9). otSSB70C had a negligible effect on the ability of human SSB to stimulate polcv activity. Combi- nation of the various antibodies did not fully abolish the enhancement of pola activity by SSB (data not shown). The basal level of polol activity, in the absence of SSB, was not affected by the antibodies.

CYSSB~OC Inhibits the Stimulation of PO16 by Human SSB- Human SSB was also found to stimulate ~016 about 20-fold in the presence of ~016 accessory factors (PCNA and the inhibitor/activator I fraction). In contrast to the situation with pola, aSSB70C blocked the stimulation of ~016 by SSB by 65% (Fig. 10). Po16 stimulation was not markedly affected by &SB70A, -7OB, and -34A. None of the antibodies affected the low levels of ~016 activity obtained in the absence of SSB.

DISCUSSION

The multisubunit nature of human SSB suggests that it may have multiple roles in DNA replication and perhaps in recombination and repair as well. One activity of the human SSB is its ability to bind with high affinity to single-stranded DNA (Wobbe et al., 1987; Fairman and Stillman, 1988; Wold and Kelly, 1988; Wold et al., 1989). Using two separate meth- ods, UV cross-linking and isolation of individual subunits, we have demonstrated that the 70-kDa polypeptide is the only subunit which binds to DNA and that the 34- and 11-kDa subunits are not required for this binding. The identification of the 70-kDa polypeptide as the DNA binding subunit has also been demonstrated by blotting the separated SSB sub- units onto nitrocellulose and probing with labeled single- stranded DNA (Wold et al., 1989).5

’ S.-H. Lee and J. Hurwitz, unpublished observations.

A. Ab: 70A 34A 70B 7oc

u I I I I I

01234567 Ab Added (pg)

Frc. 8. Effect of &SB monoclonal antibodies on SV40 DNA unwinding. Reaction mixtures (30 ~1) contained 40 mM creatine phosphate-diTris salt, pH 7.7, 7 mM MgC12, 0.5 mM dithiothreitol, 4 mM ATP, 1 fig of creatine phosphokinase, 180 ng of relaxed pSVOlAEP DNA (form I’), 5 Gg of BSA, 1.0 pg of TAg, 750 units of topo I, 1.0 pg of human SSB, and purified aSSB monoclonal antibody (Ab) as indicated. Reactions were incubated at 0 “C for 30 min and then at 37 “C for 60 min. A, samples were processed as previously described (Kenny et al., 1989) and electrophoresed at 20 V for 12 h on a 1.5% agarose gel in 1 x TAE buffer containing 1.5 fig/ml chloroquine. Gels were soaked in 50 mM NaCl for 60 min and then in 0.5 yg/ml ethidium bromide for 60 min before visualizing DNA with a UV transilluminator. Form II, nicked circular duplex DNA; form I’, relaxed circular duplex DNA; form U, highly unwound circular duplex DNA. B, reaction products were quantitated by using a laser densitometer to scan a negative photograph of the gel. The percentage of DNA molecules unwound was calculated as follows: Form U produced (%) = [form U/(form U + form I’)] X 100. Form II cannot be converted to form U in the absence of DNA ligase, and thus it was not figured into the calculation. 0, aSSB70A; 0, otSSB34A; W, aSSB70B; 0, aSSB70C.

Human SSB appears to have multiple functions in SV40 DNA replication in addition to simply binding to single- stranded DNA. These other functions include the ability to stabilize single-stranded DNA in the SV40 DNA unwinding reaction and the ability to stimulate pola and ~016. Table III summarizes the effects of the aSSB monoclonal antibodies on reactions involving human SSB. The inhibition of these reactions by the aSSB antibodies strengthens the belief that these are important functions of SSB in SV40 DNA replica- tion. It is unclear why the stimulation of polcv was only partially blocked (50-60%) by (YSSB~OA, -34A, and -7OB. Perhaps the inhibition of pola stimulation is more pro- nounced in the context of replication, or alternatively there

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Role of Human SSB in SV40 DNA Replication

FIG. 9. Effect of &SB monoclonal antibodies (Ab) on the stimulation of pola by human SSB. Reaction mixtures (27.5 ~1) contained 40 mM creatine phosphate-diTris salt, pH 7.7,7 mM MgC12, 0.5 mM dithiothreitol, 4 mM ATP, 1 rg of creatine phosphokinase, 20 pM [“H]dTTP (400 cpm/pmol), 5 Kg of BSA, and 0.25 pg of human SSB and purified antibody as indicated. Following incubation for 30 min at 0 “C, 0.1 rg (10 pM as nucleotide) poly(dA)4000.01igo(dT)ln~,a (20~1, w/w) and 0.05 unit of polol-primase were added to give a final reaction volume of 30 ~1. Reactions were incubated for 60 min at 37 “C, and acid-insoluble radioactivity was determined. Open symbols, no SSB added; closed symbols, human SSB added; 0, 0, aSSB70A; W, 0, olSSB34A; A, A, aSSB70B; V, V, &SB70C.

FIG. 10. Effect of &SB monoclonal antibodies (Ab) on the stimulation of ~016 by human SSB. Reaction mixtures (22 pl) contained 40 mM creatine phosphate-diTris salt, pH 7.7,7 mM MgCl2, 0.5 mM dithiothreitol, 4 mM ATP, 1 pg of creatine phosphokinase, 20 PM [“H]dTTP (400 cpm/pmol), 5 pg of BSA, and 0.5 pg of human SSB and purified antibody as indicated. Following incubation for 30 min at 0 “C, 0.1 pg (10 FM as nucleotide) poly(dA)rooo.oligo(dT)12~1R (20:1, w/w), 0.1 unit of polb, 0.2 pg of PCNA, and 1.5 fig of inhibitor/ activator I fraction were added to give a final reaction volume of 30 ~1. Reactions were incubated for 60 min at 37 “C, and acid-insoluble radioactivity determined. Open symbols, no SSB added; closed sym- bols, human SSB added; 0, 0, &SB70A; W, 0, olSSB34A; A, a, nSSB70B; v, V, nSSB70C.

may be another as yet unidentified function of SSB which is also affected by these antibodies. It should be noted that aSSB70A, -34A, and -7OB each inhibited SV40 DNA repli- cation by more than 70%, although pola has been thought to be responsible only for lagging strand synthesis. This may be consistent with another function of SSB, or alternatively, pola and SSB may be involved in the initiation of leading strand synthesis. In fact, it has been shown recently that ~016 does not efficiently utilize RNA primers, suggesting that polol may be required for the initial stages of leading strand syn- thesis (Lee et al., 1989a).

Although &SB70A, -34A, and -7OB blocked the stimula- tion of pola, aSSB70C inhibited DNA unwinding and ~016 stimulation. This is consistent with the observation that various viral and prokaryotic SSBs stimulate ~016 and func- tion in the unwinding reaction, but the stimulation of pola is specific to human SSB (Kenny et al., 1989). Thus, the stim-

TABLE III Summary of the effects of nSSB monoclonal antibodies

The effect of &SB monoclonal antibodies (Ab) on reactions in- volving human SSB is summarized. + indicates that at least 75% of the activity remained in the presence of the antibody. - indicates that less than 50% of the activity remained in the presence of the antibody.

Activity in the presence of Ab

Ab DNA DNA DNA Pola PO16 added replication binding unwinding stimulation stimulation

70A - + + - + 34A - + + - + 70B - + + - + 7oc - + - + -

ulatory effects of human SSB on pola and ~016 are likely to occur by different mechanisms. Preliminary results suggest that human SSB may increase the processivity of pola. In contrast, the initiation frequency of ~016 may be affected.4 The specificity of polo! stimulation by human SSB suggests that there may be a direct protein-protein interaction. The monoclonal antibodies described in this paper should prove useful in testing this possibility.

None of the &SB antibodies inhibited the binding of human SSB to single-stranded DNA as assayed by nitrocel- lulose filter binding. However, it is possible that DNA binding may be altered in some manner. For example, the antibodies could affect the ability of SSB to bind cooperatively or to stabilize single-stranded DNA. Three of the antibodies (&SB70A, -34A, and -7OB) formed a tertiary complex (anti- body. SSB . DNA) as detected by immunoprecipitation and gel mobility shift assays. No tertiary complex was detected be- tween aSSB70C, human SSB, and DNA. This result was somewhat unexpected, since aSSB70C clearly binds to SSB and did not inhibit the binding of SSB to DNA. Perhaps a metastable tertiary complex does form but is disrupted by the immunoprecipitation or electrophoresis procedures.

Since the olSSB antibodies recognize two different SSB subunits, inferences can be drawn as to the functions of the individual subunits. In addition to being necessary and suffi- cient for DNA binding, the 70-kDa subunit contains separate epitopes involved in DNA unwinding and stimulation of pola and ~016. The 34-kDa subunit also appears to be involved in the stimulation of pola activity. The function of the 11-kDa subunit is not known at present. These data suggest the existence of at least three separate functional domains: 1) one domain required for DNA binding that is unaffected by the aSSB monoclonal antibodies, 2) a second domain recognized by aSSB70C and required for DNA unwinding and ~016 stimulation, and 3) a third domain recognized by aSSB70A, -34A, and -7OB that is involved in the stimulation of pola. The cvSSB monoclonal antibodies described in this report should continue to prove useful in the study of the molecular biology and biochemical functions of human SSB.

Acknowledgments-We thank Nilda Belgado and Barbara Phillips for technical assistance. We appreciate the help of Drs. Monika Lusky and Lilian Clark with the immunoblotting experiments. We also thank Suk-Hee Lee, Takashi Matsumoto, Peter Bullock, and Toshih- iko Eki for providing some of the enzymes used in this work. We are grateful to Drs. Frank Dean and Jim Borowiec for helpful comments on this manuscript.

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M K Kenny, U Schlegel, H Furneaux and J Hurwitzsubunits in simian virus 40 DNA replication.

The role of human single-stranded DNA binding protein and its individual

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