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- 1989; Challberg and Kelly, 1989; Hay and Russell, 1989).
man SSB) is a multisubunit protein containing poly- However, the sole viral protein required for replication, SV40
peptides of 70, 34, and 11 kDa that is required for large tumor antigen (TAg), provides multiple functions in-
SV40 DNA replication in vitro. In this report we iden- cluding origin binding and melting and DNA helicase activi-
tify the functions of the SSB and its individual subunits ties (Tjian, 1978; Deb and Tegtmeyer, 1987; Borowiec and
in SV40 DNA replication. The 70 kDa subunit was Hurwitz, 1988a, 198813; Stahl et al., 1986). In the presence of
found to bind to single-stranded ‘DNA, whereas the ATP at 37 “C, TAg forms a bilobed, nucleoprotein structure
other subunits did not. Four monoclonal antibodies at the SV40 origin of replication in which the origin DNA is
against human SSB were isolated which inhibited
distorted and locally melted (Dean et al., 1987c; Mastrangelo
SV40 DNA replication in vitro. The antibodies have

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et al., 1989; Borowiec and Hurwitz, 1988b; Roberts, 1989;
been designated aSSB70A, aSSB70B, aSSB70C, and
Dean et al., 1989). The addition of human SSB and topoisom-
aSSB34A to indicate which subunits are recognized.
Immunolocalization experiments indicated that human erase I or II leads to extensive unwinding of the DNA (Dean
SSB is a nuclear protein. Human SSB is required for et al., 1987a, 1987b; Dodson et al., 1987; Wold et al., 1987),
the SV40 large tumor antigen-catalyzed unwinding of although this unwinding may normally be coupled to DNA
SV40 DNA and stimulates DNA polymerases (pol) a synthesis. Subsequent events in SV40 DNA replication are
and 6. The DNA unwinding reaction and stimulation of unclear, but several proteins have been implied to play specific
~016 were blocked by aSSB70C, whereas the stimula- roles. DNA polymerase a-primase complex (pola-primase) is
tion of pola! by human SSB was unaffected by this likely to be involved in the initiation reaction and lagging
antibody. Conversely, aSSB70A, -7OB, and -34A in- strand synthesis, whereas DNA polymerase 6 (pals), prolifer-
hibited the stimulation of pola, but they had no effect ating cell nuclear antigen (PCNA), and other auxilliary fac-
on DNA unwinding and ~016 stimulation. None of the tors have been implicated in leading strand synthesis (Murak-
antibodies inhibited the binding of SSB to single- ami et al., 1986, Prelich et al., 1987; Prelich and Stillman,
stranded DNA. These results suggest that DNA un- 1988; Downey et al., 1988; Lee et al., 1989a, 1989b; Wold et
winding and stimulation of pola and ~016 are required al., 1989; Tsurimoto and Stillman, 1989). RNA primers can
functions of human SSB in SV40 DNA replication. The
be removed by the combined action of RNase H and 5’ + 3’
human SSB ‘IO-kDa subunit appears to be required for
exonuclease. After gap-filling by one of the polymerases, DNA
DNA unwinding and ~016 stimulation, whereas both
the 70- and 34-kDa subunits may be involved in the ligase can seal nicks, and topoisomerase II can decantenate
stimulation of pola. 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
The in vitro SV40 DNA replication system has provided an report, the role of human SSB and its individual subunits in
assay for the identification and isolation of a single-stranded
these reactions and in SV40 DNA replication has been inves-
DNA binding protein from human cells (human SSB)l which tigated using anti-SSB monoclonal antibodies and other tech-
is analogous to the SSBs from Escherichia coli and bacterio-
niques.
phage T4 (T4 gene 32 protein) (Wobbe et al., 1987; Wold and
Kelly, 1988; Fairman and Stillman, 1988). The role of human MATERIALS AND METHODS
SSB in SV40 DNA replication, and perhaps by analogy,
Reagents-The following reagents were obtained commercially:
cellular DNA replication, is now being investigated. p~ly(dA)~~~ (Life Sciences); micrococcal nuclease, E. coli SSB, T4g32,
SV40 depends almost entirely on the host cell to provide Sephadex G-25, and ohgo(dT), (Pharmacia LKB Biotechnology
its replication machinery (for recent reviews, see Stillman, Inc.); radionucleotides (Du Pont-New England Nuclear); unlabeled
nucleotides, M13mp18 DNA, Ml3 sequencing primer (dTCCCAG-
* This work was supported by Grant GM34559 from the National TCACGACGT), DNase I, and E. coli DNA polymerase I (Boehringer
Institutes of Health. The costs of publication of this article were Mannheim); creatine phosphokinase (Worthington); chloroquine
defrayed in part by the payment of page charges. This article must (Sterling Drug Inc.); protein A-Sepharose (Sigma); goat anti-mouse
therefore be hereby marked “aduertisement” in accordance with 18 agarose (HyClone); immunoblotting reagents (Bio-Rad); immunohis-
U.S.C. Section 1734 solely to indicate this fact. tochemical reagents (Vector Laboratories). Bovine serum albumin
1 The abbreviations used are: SSB, single-stranded DNA binding (BSA, Miles Laboratories) was heat-denatured before use. TAE buffer
protein; SV40, simian virus 40; TAg, SV40 large tumor antigen; pola, (1 X) is 40 mM Tris acetate and 1 mM EDTA.
DNA polymerase ol; polb, DNA polymerase 6; PCNA, proliferating DNA and Replication Proteins-The SV40 origin-containing plas-
cell nuclear antigen; BSA, bovine serum albumin; T4g32, bacterio- mid pSVOlAEP has been described previously (Wobbe et al., 1985).
phage T4 gene 32 protein; IgG, immunoglobulin G; BrdUrd, bromo- Crude extract (Wobbe et aZ., 1985), topo I (Ishimi et al., 1988), pol~l-
deoxyuridine. primase (Ishimi et al., 1988), PCNA-dependent ~016 (Lee et al., 1989b),

7693
7694 Role of Human SSB in SV40 DNA Replication
PCNA (Lee et a!., 1988), and the 0.4 M double-stranded DNA cellulose DNA Binding Assay-Nitrocellulose filters were pretreated with
inhibitor/activator I fraction (Lee et a&, 1989a) were prepared from alkali to reduce nonspecific binding of DNA which was not protein-
HeLa cells as described elsewhere. TAg was immunoaffinity-purified mediated (McEntee et al., 1980). DNA binding reactions (50 pl),
(Mastrangelo et al., 1989) from insect cells infected with a TAg- containing 50 mM Tris-HCl, pH 7.5, 5 mM MgCl*, 0.5 mM dithiothre-
encoding, recombinant baculovirus vector (O’Reilly and Miller, 1988). itol, 0.2 mg/ml BSA, 4 ng of single-stranded [32P]DNA, and HeLa
Adenovirus DNA binding protein was purified from adenovirus- SSB or isolated subunits, were incubated for 10 min at 37 “C. Mix-
infected HeLa cells (Ikeda et al., 1981). tures were filtered through nitrocellulose filters and washed with 1
Human SSB was isolated from 1.2 x 10” HeLa cells by a modifi- ml of 50 mM Tris-HCl, pH 7.5, 5 mM MgCl,. Radioactivity retained
cation of the procedure of Wobbe et al. (1987). One unit of human on the filters was determined by liquid scintillation spectroscopy.
SSB supported the incorporation of 1 nmol of dTMP in 60 min in Mono&ma1 Antibodies-Mouse hybridomas were developed com-
the SV40 DNA replication assay (Wobbe et al., 1987). HeLa crude mercially by American Biotechnologies Inc. using purified human
extract (9.6 g of protein, 6600 units, 600 ml) was fractionated by O- SSB as the antigen. Hybridomas were grown in serum-free media,
35% ammonium sulfate precipitation as described previously (Wobbe and the supernatant was collected. The isotype of all four antibodies
et al., 1987). The AS35 fraction (2.9 g of protein, 5900 units, 144 ml) characterized here was found to be IgGlk, although some in uitro
was adjusted to 0.5 M NaCl in buffer BN (20 mM Tris-HCl, pH 7.5, class switching was observed. Hybridoma supernatant was adjusted
10% glycerol, 0.1 mM EDTA, 0.01% Nonidet P-40, 1 mM dithiothre- to 50 mM Tris-HCl, pH 8.5, 3 M NaCl and loaded onto a protein A-
itol) and filtered through filter paper (Whatman 2). This fraction was Sepharose column. The column was washed with 10 mM Tris-HCl,
loaded onto a 6-ml (-0.5 g of protein/ml bed volume) single-stranded pH 8.5, 3 M NaCl, and antibodies were eluted with 100 mM glycine
DNA cellulose column (3 mg of DNA/g of cellulose, Sigma) equili- HCl, pH 2.5. Purified antibodies were neutralized and dialyzed against
brated with buffer BN containing 0.5 M NaCl. A short, squat column phosphate-buffered saline and stored in small aliquots at -80 “C.
(2.5 X 10 cm, Kontes) was used to prevent clogging such a small Zmmunohistochemical Analysis-Cultured human cells (HEp-2)
amount of resin with the crude fraction. The column was washed fixed on slides (Behring Diagnostics Inc.) were incubated with 2 pg/
extensively (-10 bed volumes) with buffer BN containing 0.5 M NaCl. ml purified monoclonal antibody for 18 h at 4 “C. After washing with
Active fractions were eluted with buffer BN containing 2 M NaCl. phosphate-buffered saline, antibody binding was detected by the
The SSB (3.7 mg protein, 2400 units, 5.8 ml), which was >80% pure sequential application of biotinylated horse anti-mouse secondary
at this stage, was diluted with buffer BN to 50.1 mg protein/ml and antibody and avidin-biotin peroxidase complex (Hsu et al., 1981).

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550 mM NaCl and applied to a 2-ml phosphocellulose column equil- Peroxidase was visualized by incubation with diaminobenzidine and
ibrated with buffer BN containing 50 mM NaCl. After washing with photographed using phase contrast microscopy.
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 RESULTS
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) The Structure of Human SSB-Human SSB (also referred
containing 0.25 M NaCl and stored at -80 “C (1.8 mg of protein, 1300 to as HeLa SSB, RF-A, and RP-A) has been purified from
units, 1.1 ml). Recently, a fast protein liquid chromatography Mono
HeLa and 293 cells based on its ability to support SV40 DNA
Q column was substituted for the phosphocellulose column in order
to remove contamination by a small amount of nuclease activity replication in an in uitro eomplementation assay (Wobbe et
which inhibited replication reactions containing purified proteins. al., 1987; Fairman and Stillman, 1988; Wold and Kelly, 1988).
The Mono Q column was loaded and washed with 0.1 M NaCl in Based on the recovery of SSB replication activity from crude
buffer BN and was eluted with a linear gradient from 0.1 to 0.5 M extracts, a minimum of 50,000 SSB molecules/HeLa cell is
NaCl in the same buffer. The SSB eluted at 0.26 M NaCl. estimated. This number may be an underestimate if only a
The key feature of this purification procedure was the loading of fraction of the SSB was extracted from the cell. As shown in
the single-stranded DNA cellulose column in high salt which resulted
in >99.8% of the protein flowing through the column and >300-fold Fig. 1 and previously (Fairman and Stillman, 1988; Wold and
purification. Previously, a double-stranded DNA cellulose column has Kelly, 1988), SSB purified to apparent homogeneity from
been used (Wobbe et al., 1987), hut it was found to have a limited human cells consists of 3 tightly associated polypeptides of
SSB binding capacity, suggesting that the SSB may have bound to approximately 70, 34, and 11 kDa. The largest subunit mi-
regions of single-stranded DNA on the resin. SSB-containing frac- grated as a smear or two to three distinct bands depending on
tions could be pooled based on protein determinations rather than the resolution of the SDS-PAGE and regardless of the pres-
activity profiles in order to hasten the procedure. Concentrated
preparations of human SSB were found to precipitate out of solution ence of reducing agents (Fig. 1; Wobbe et al., 1987; and data
in low salt and thus were stored in 0.25 M NaCl. This purification not shown). The “70-kDa bands” are immunologically related
procedure was rapid and reproducible and resulted in relatively pure, to each other and may represent post-translational modifica-
concentrated, and soluble SSB. tions. In contrast, the 70-, 34-, and ll-kDa subunits are
Labeled DNAs-“P-Labeled DNA was prepared by primer exten- antigenically distinct (data not shown). Human SSB has an
sion using Ml3 as a template. Reaction mixtures (50 ~1) contained
isoelectric point of 5.6.’ Gel filtration chromatography and
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 glycerol gradient centrifugation of human S$B indicated that
mer), 40 pM each of dATP, dGTP, dTTP, and [a-32P]dCTP (300 the protein has a Stokes radius of about 52 A and a sedimen-
PCi), and 5 units of E. coli DNA polymerase I. When the DNA was tation coefficient of approximately 5.1 S (Wobbe et al., 1987;
used for UV cross-linking experiments, bromo-dUTP was substituted Fairman and Stillman, 1988; Wold and Kelly, 1988; and data
for dTTP. Reactions were incubated for 30 min at 30 “C followed by not shown). Assuming a partial specific volume of 0.725 cm3/
isolation of the DNA by phenol/chloroform extraction and Sephadex g, a native molecular mass of 110 kDa and a frictional ratio
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. of 1.65 can be calculated by the method of Siegel and Monty
CJV Cross-linking-UV cross-linking reactions (50 ~1) containing (1966). These data are consistent with SSB having a some-
50 mM Tris-HCl, pH 7.5, 5 mM MgCl,, 0.5 mM dithiothreitol, 0.2 mg/ what prolate structure and a subunit stoichiometry of 1:l:l.
ml BSA, 4 ng of single-stranded 32P-labeled BrdUrd-DNA, and 100 Further studies will be required to better define the structure
ng of the indicated SSB were incubated at 37 “C for 10 min. Reaction of human SSB. Human SSB was observed to aggregate at a
mixtures were spotted on Saran Wrap on top of a Photoproducts low ionic strength and high SSB concentrations. Other SSBs
TM-36 mid-range UV transilluminator (302 nm; 8000 pW/cm*) and
illuminated for 4 min at room temperature. Mixtures were then also have the tendency to aggregate, and this property may
transferred to 1.5-ml Eppendorf test tubes, and 1.0 pg of DNase I, 5 be related to the cooperative manner in which SSBs usually
units of micrococcal nuclease and CaClz to 1.5 mM were added. bind to DNA (Chase and Williams, 1986; Williams and Chase,
Reactions were incubated at 37 “C for 20 min and stopped by addition 1989). The mechanism by which human SSB binds to DNA
of 50 ~1 of a solution containing 180 mM sodium pyrophosphate and is not well characterized.
50 mM EDTA. Proteins were precipitated with 10% trichloroacetic
acid, washed with ether, and resuspended in SDS-PAGE sample
buffer. 2 G. Hodgins and J. Hurwitz, unpublished observations.
 Role of Human SSB in SV40 DNA Replication

top- . top -
200-
93 - Ad DBP, 200 -
rm
45 - =HSSB-70
66 -

45 -
.o - T4g32 93 -

- HSSB-34 Y
- -34 31 -
1 Ad DBP,
31 - HSSB-70
66 -
e
22 - -E. SSB

45 -
14 -
- HSSB-11 0
22 - - T4g32
front - 0
front -

1234 1234
FIG. 2. UV cross-linking of SSBs to “P, BrdUrd-DNA. The
14-w -11

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indicated SSBs were incubated with ‘“P-labeled, BrdUrd-substituted
single-stranded DNA and exposed to UV light as described under
FIG. 1. SDS-polyacrylamide gel electrophoresis of human “Materials and Methods.” After nuclease treatment, the SSBs were
SSB. Electrophoresis of 0.5 rg of human SSB was carried out electrophoresed on SDS-polyacrylamide gels followed by autoradiog-
according to the method of Laemmli (1970) using a 4% stacking gel raphy. A, 16% SDS-PAGE. B, 10% SDS-PAGE. Molecular mass
and a 14% separating gel. The 70-, 34-, and 11-kDa subunits are markers are indicated on the left in kilodaltons. The positions of
labeled on the right. Molecular mass markers are indicated on the unlabeled SSBs are indicated on the right. Note that SSBs cross-
left: phosphorylase b (93 kDa), bovine serum albumin (66 kDa), linked to short DNA fragments migrate slightly slower than free
ovalbumin (45 kDa), carbonic anhydrase (31 kDa), soybean trypsin SSBs. HSSB-70, -34, and -II denote the human SSB 70-, 34., and
inhibitor (22 kDa), lysozyme (14 kDa). The gel was silver-stained 11-kDa subunits, respectively. Ad DBP, adenovirus DNA binding
using a kit from ICN Biochemicals. proteins.

The Human SSB 70-kDa Subunit Binds to Single-stranded 50 -

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- 11 ho0
fg34kOO
otide complexes were then electrophoresed on SDS-polyacryl- 0 I
0 1 2 3 4
amide gels followed by autoradiography (Fig. 2). Only the 70- HeLa SSB Fraction(~ll
kDa subunit of human SSB was labeled by this method,
indicating that it was bound to the DNA, whereas the 34- and FIG. 3. DNA binding activity of the isolated human SSB
subunits. The 70-, 34-, and 11-kDa subunits of the human SSB were
ll-kDa subunits were not in direct contact with the DNA. separated by SDS-PAGE, extracted from gel slices, and renatured
The adenovirus DNA binding protein (adenovirus DNA bind- separately or in combination by the procedure of Hager and Burgess
ing protein, 72 kDa) and the T4 gene 32 protein (T4g32, 33.5 (1980). The SSB subunits were tested for their ability to bind single-
kDa) yielded labeled bands of the appropriate sizes. However, stranded IR’P]DNA in a nitrocellulose filter binding assay as described
the rate of migration of the proteins was somewhat reduced under “Materials and Methods.” The concentration of the isolated
subunits could not be directly determined because of the presence of
by the attached DNA. The 19-kDa E. coli SSB, which binds
bovine serum albumin in the buffers. However, based on SDS-poly-
to DNA as a tetramer, produced a more complex series of acrylamide gel analysis, there was approximately equal recovery of
bands corresponding to one or more protomers of the SSB. each of the subunits. Thus, equal volumes of the isolated human SSB
Although the results presented in Fig. 2 indicate that the subunits contained the same ratio of polypeptides as found in the
human SSB 70-kDa subunit binds to DNA, it is possible that intact SSB protein. The SSB subunits assayed were as indicated: 0,
the other subunits may be required for this activity. To test 70-, 34-, and ll-kDa subunits; 0, ‘IO-kDa subunit; n , 34.kDa subunit;
0, 11-kDa subunit.
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 the presence or absence of the other subunits. The 34- and
subunits were then tested for their ability to bind single- 11-kDa subunits showed little or no DNA binding activity,
stranded DNA using a nitrocellulose filter binding assay (Fig. and they did not significantly alter the ability of the 70-kDa
3). The 70-kDa subunit exhibited DNA binding activity in subunit to bind DNA. We have thus far been unable to
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-

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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
sections or the use of bright field microscopy also indicated
kL
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
FIG. 5. Cellular localization of human SSB. HEp-2 cells were
of (YSSB~OA and -7OB antibodies inhibited replication, but a incubated with &SB’IOA (A), nSSB70C (B), &SB34A (C), or non-
maximum of 75% inhibition was observed. The effect of immune mouse IgG (D) and visualized by peroxidase staining and
cuSSB34 plateaued at 85% inhibition, while aSSB70C de- phase contrast microscopy. Note that some cells have apparently
creased nucleotide incorporated by more than 95% at high fused to form syncytia and thus are multinucleated.

1234

I
70- -

34- I

Ab Added

FIG. 6. Inhibition of SV40 DNA replication by aSSB mono-

11 - 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
FIG. 4. Identification of human SSB subunits recognized by phosphate-diTris salt, pH 7.7, 7 mM MgCl,, 0.5 mM dithiothreitol, 4
aSSB monoclonal antibodies. Purified human SSB was subjected mM ATP, 200 pM each of UTP, GTP, and CTP, 100 WM each of
to SDS-PAGE on a 14% gel and then transferred to nitrocellulose by dATP, dGTP, and dCTP, 20 pM [“H]dTTP (300 cpm/pmol), 1 pg of
electroblotting. Nitrocellulose strips were incubated sequentially with creatine phosphokinase, 180 ng of pSVOlAEP DNA, 10 pg of BSA,
&SB hybridoma supernatant, alkaline phosphatase-conjugated goat and 1.0 tig of TAR. Following incubation at 37 “C for 60 min. acid-
anti-mouse antibody, and alkaline phosphatase color development insoluble radioac&ity was determined. 0, olSSB70A; 0, (uSSB34A;
reagents. Lane 1, &SB70A; lane 2, uSSB70B; lane 3, aSSB70C; lane W, nSSB70B; 0, &SB70C; A, equimolar combination of c&SB34A,
4, olSSB34A. -7OB, and -70C (totaling the indicated value).
 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,
. 706
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 P I I I I , I
SV40 DNA replication by the cvSSB antibodies was caused 0 1 2 3 4 5 6
Human SSB Added trg)
specifically by the neutralization of human SSB in the crude
extract.3 FIG. 7. Reconstitution of inhibited replication reactions by
addition of purified human SSB. HeLa crude extract (170 pg of
The Antibodies Do Not Inhibit DNA Binding-The effect
protein) and purified monoclonal antibodies (Ab) (0.45 pg) were
of aSSB monoclonal antibodies on the binding of human SSB incubated with various amounts of human SSB for 30 min at 0 “C in
to 32P-labeled, single-stranded DNA was tested using a nitro- a volume of 15.5 pl. The remaining reaction components were added
cellulose filter binding assay. In the absence of SSB the DNA as in Fig. 6, and reaction mixtures were incubated for 60 min at 37 “C.
passed through the filter, whereas in the presence of limiting V, no antibody added; 0, crSSB70A; 0, olSSB34A; n , otSSB70B; Cl,
levels of human SSB approximately 25% of the DNA was olSSB70C; A, combination (comb) of olSSB34A, -7OB, and -70C (0.15
fig of each).
retained (Table I). None of the antibodies inhibited the bind-
ing of SSB to DNA, and the antibodies alone, in the absence

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of SSB, did not exhibit any DNA binding activity. Higher TABLE I
levels of antibodies (up to 3 pg of antibody:1 ng of SSB) did Effect of LYSSB monoclonal antibodies on the binding of human SSB
to single-stranded DNA
not affect DNA binding (data not shown). Although the 70-
kDa SSB subunit binds to DNA, &SB70A, -7OB, and -70C The effect of antibodies on the binding of SSB to single-stranded
[32P]DNA was tested using a nitrocellulose filter binding assay. Re-
antibodies must react with epitopes which are not required action mixtures (49 ~1) contained 50 mM Tris-HCl, pH 7.5, 50 mM
for DNA binding. NaCl, 5 mM MgCl,, 0.5 mM dithiothreitol, 10 pg of BSA, and 1.0 ng
Since the aSSB monoclonal antibodies specifically recog- of human SSB and 30 ng of purified monoclonal antibody as indicated.
nize SSB but do not inhibit DNA binding, it is possible that Reactions were incubated at 0 “C for 30 min followed by addition of
an antibody. SSB . DNA complex can form. The formation of (1 ~1) 12 pmol (as nucleotide) of 32P-labeled, single-stranded DNA
a tertiary complex between &SB antibodies, SSB, and 32P- and further incubation at 37 “C! for 10 min. Reaction mixtures were
filtered on nitrocellulose filters and washed with 1 ml of a solution
labeled, single-stranded DNA was tested by immunoprecipi- containing 50 mM Tris-HCl, pH 7.5, 50 mM NaCl, and 5 mM MgClz
tation with goat anti-mouse IgG (Table II). Levels of nonspe- and radioactivity retained on the filters was determined. The amount
cific binding (i.e., in the absence of SSB or antibody) were of SSB used in these experiments was in the linear assav range.
Human SSB Antibody
3 In the experiments described in Figs. 6 and 7, crude extracts were DNA retained
added added
first preincubated with the antibodies at 0 “C followed by the addition
pm01
of reagents essential for the replication reaction. The addition of
large amounts of human SSB partially reversed the effects of anti- - co.3
bodies 70B and 34A (Fig. 7). At the level of antibody used (0.45 pg), - 2.8
the addition of human SSB prior to the addition of crude extracts 70A 3.0
did not alter the results described in Fig. 7. High levels of SSB only 34A 2.9
partially reversed the effects of antibodies 70B and 34A. These results 70B 3.2
could be due to the presence of nonspecific inhibitors in these two 7oc 3.2
antibody preparations. We consider this unlikely for the following 70A.34A.70B.m 70C co.3
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 significant but relatively constant. A tertiary complex was
of the antibodies with increasing concentrations of human SSB clearly formed when &SB70A, -34A, or -7OB was used. Sur-
reversed the inhibition. Thus when 0.2 pg of antibody 34A was prisingly, no complex was detected between &SB70C, SSB,
preincubated for 15 min at 0 “C, with 0, 0.7, and 1.5 rg of human and DNA. Increasing the level of &SB70C or altering the
SSB, followed by incubation with crude extracts for 30 min at 0 “C,
order of addition of antibody, SSB, and DNA did not change
replication was inhibited 82, 40, and 23%, respectively; when the
antibody was preincubated with 3 pg of human SSB, the replication this result. Similar results were obtained using a gel mobility
reaction was stimulated 32%. As noted in Fig. 7, we have consistently shift assay.“ A slow-migrating complex between SSB and
observed the replication reactions with crude extracts can be stimu- single-stranded DNA was detected, and the migration of this
lated by supplementation with human SSB. Similar results were complex was further retarded by aSSB70A, -34A, and -7OB.
obtained with the antibody 70B. High levels of antibodies (1 pg and However, aSSB70C had no detectable effect on the migration
higher in some cases) did not affect a number of individual enzymatic
reactions examined. Thus the human SSB-dependent DNA unwind- of the SSB . DNA complex. This suggests that this antibody
ing reaction catalyzed by T antigen and the SSB-stimulated elonga- neither inhibited DNA binding nor did it stably bind to the
tion of primed DNA templates by DNA polymerases 01 and 6, when SSB . DNA complex.
affected, required high concentrations of antibodies. This suggests aSSB70C Inhibits SV40 DNA Unwinding-The effect of
that the SSB-antibody complexes were capable of participating in c&SB antibodies on unwinding was tested in reactions con-
these individual reactions. If each reaction (unwinding, elongation, taining TAg, human SSB, topoisomerase I, and SV40 origin-
etc.) were partially inhibited, the product of these individual effects
could be greater than the sum of each effect. Since all of these containing DNA (Fig. 8). DNA unwinding was virtually abol-
reactions are essential for SV40 DNA replication, this would explain ished by &SB70C, whereas the other antibodies had little or
the more pronounced inhibitory effect of low levels of these antibodies no effect. The ratio of otSSB70C antibody to SSB required to
on the overall replication reaction than their effect on individual
reactions. 4 L. Clark and J. Hurwitz, unpublished observations.
7698 Role of Human SSB in SV40 DNA Replication
TABLE II
A.
Immunoprecipitation of nSSB antibody. SSB. DNA complexes
Ab: 70A 34A 70B 7oc
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 Human SSB
DNA precipitated
added added
pm01
70A 1.8
34A 2.0
70B 2.0
7oc 2.1
- 1.5
70A 5.0

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34A 5.3
70B 7.2
7oc 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 u
polcv approximately lo-fold using poly(dA) oligo(dT) as a I I I I I
template under the same conditions used for DNA replication 01234567
and unwinding reactions. The monoclonal antibodies Ab Added (pg)
aSSB70A, -7OB, and -34A blocked the stimulation of polcv by
Frc. 8. Effect of &SB monoclonal antibodies on SV40 DNA
SSB by 50-65% (Fig. 9). otSSB70C had a negligible effect on unwinding. Reaction mixtures (30 ~1) contained 40 mM creatine
the ability of human SSB to stimulate polcv activity. Combi- phosphate-diTris salt, pH 7.7, 7 mM MgC12, 0.5 mM dithiothreitol, 4
nation of the various antibodies did not fully abolish the mM ATP, 1 fig of creatine phosphokinase, 180 ng of relaxed
enhancement of pola activity by SSB (data not shown). The pSVOlAEP DNA (form I’), 5 Gg of BSA, 1.0 pg of TAg, 750 units of
basal level of polol activity, in the absence of SSB, was not topo I, 1.0 pg of human SSB, and purified aSSB monoclonal antibody
affected by the antibodies. (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
CYSSB~OC Inhibits the Stimulation of PO16 by Human SSB-
described (Kenny et al., 1989) and electrophoresed at 20 V for 12 h
Human SSB was also found to stimulate ~016 about 20-fold on a 1.5% agarose gel in 1 x TAE buffer containing 1.5 fig/ml
in the presence of ~016 accessory factors (PCNA and the chloroquine. Gels were soaked in 50 mM NaCl for 60 min and then
inhibitor/activator I fraction). In contrast to the situation in 0.5 yg/ml ethidium bromide for 60 min before visualizing DNA
with pola, aSSB70C blocked the stimulation of ~016 by SSB with a UV transilluminator. Form II, nicked circular duplex DNA;
by 65% (Fig. 10). Po16 stimulation was not markedly affected form I’, relaxed circular duplex DNA; form U, highly unwound
circular duplex DNA. B, reaction products were quantitated by using
by &SB70A, -7OB, and -34A. None of the antibodies affected a laser densitometer to scan a negative photograph of the gel. The
the low levels of ~016 activity obtained in the absence of SSB. percentage of DNA molecules unwound was calculated as follows:
Form U produced (%) = [form U/(form U + form I’)] X 100. Form
DISCUSSION 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,
The multisubunit nature of human SSB suggests that it otSSB34A; W, aSSB70B; 0, aSSB70C.
may have multiple roles in DNA replication and perhaps in
recombination and repair as well. One activity of the human Human SSB appears to have multiple functions in SV40
SSB is its ability to bind with high affinity to single-stranded DNA replication in addition to simply binding to single-
DNA (Wobbe et al., 1987; Fairman and Stillman, 1988; Wold
stranded DNA. These other functions include the ability to
and Kelly, 1988; Wold et al., 1989). Using two separate meth-
stabilize single-stranded DNA in the SV40 DNA unwinding
ods, UV cross-linking and isolation of individual subunits, we
reaction and the ability to stimulate pola and ~016. Table III
have demonstrated that the 70-kDa polypeptide is the only
summarizes the effects of the aSSB monoclonal antibodies
subunit which binds to DNA and that the 34- and 11-kDa
on reactions involving human SSB. The inhibition of these
subunits are not required for this binding. The identification
reactions by the aSSB antibodies strengthens the belief that
of the 70-kDa polypeptide as the DNA binding subunit has
also been demonstrated by blotting the separated SSB sub- these are important functions of SSB in SV40 DNA replica-
units onto nitrocellulose and probing with labeled single- tion. It is unclear why the stimulation of polcv was only
stranded DNA (Wold et al., 1989).5 partially blocked (50-60%) by (YSSB~OA, -34A, and -7OB.
Perhaps the inhibition of pola stimulation is more pro-
’ S.-H. Lee and J. Hurwitz, unpublished observations. nounced in the context of replication, or alternatively there
 Role of Human SSB in SV40 DNA Replication
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 - + - + -
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, ulatory effects of human SSB on pola and ~016 are likely to
0.5 mM dithiothreitol, 4 mM ATP, 1 rg of creatine phosphokinase, 20 occur by different mechanisms. Preliminary results suggest
pM [“H]dTTP (400 cpm/pmol), 5 Kg of BSA, and 0.25 pg of human that human SSB may increase the processivity of pola. In
SSB and purified antibody as indicated. Following incubation for 30
contrast, the initiation frequency of ~016 may be affected.4
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 The specificity of polo! stimulation by human SSB suggests
reaction volume of 30 ~1. Reactions were incubated for 60 min at that there may be a direct protein-protein interaction. The
37 “C, and acid-insoluble radioactivity was determined. Open symbols, monoclonal antibodies described in this paper should prove

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no SSB added; closed symbols, human SSB added; 0, 0, aSSB70A; useful in testing this possibility.
W, 0, olSSB34A; A, A, aSSB70B; V, V, &SB70C.
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.
FIG. 10. Effect of &SB monoclonal antibodies (Ab) on the Since the olSSB antibodies recognize two different SSB
stimulation of ~016 by human SSB. Reaction mixtures (22 pl) subunits, inferences can be drawn as to the functions of the
contained 40 mM creatine phosphate-diTris salt, pH 7.7,7 mM MgCl2, individual subunits. In addition to being necessary and suffi-
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 cient for DNA binding, the 70-kDa subunit contains separate
SSB and purified antibody as indicated. Following incubation for 30 epitopes involved in DNA unwinding and stimulation of pola
min at 0 “C, 0.1 pg (10 FM as nucleotide) poly(dA)rooo.oligo(dT)12~1R and ~016. The 34-kDa subunit also appears to be involved in
(20:1, w/w), 0.1 unit of polb, 0.2 pg of PCNA, and 1.5 fig of inhibitor/ the stimulation of pola activity. The function of the 11-kDa
activator I fraction were added to give a final reaction volume of 30
subunit is not known at present. These data suggest the
~1. Reactions were incubated for 60 min at 37 “C, and acid-insoluble
radioactivity determined. Open symbols, no SSB added; closed sym- existence of at least three separate functional domains: 1) one
bols, human SSB added; 0, 0, &SB70A; W, 0, olSSB34A; A, a, domain required for DNA binding that is unaffected by the
nSSB70B; v, V, nSSB70C. aSSB monoclonal antibodies, 2) a second domain recognized
by aSSB70C and required for DNA unwinding and ~016
may be another as yet unidentified function of SSB which is stimulation, and 3) a third domain recognized by aSSB70A,
also affected by these antibodies. It should be noted that -34A, and -7OB that is involved in the stimulation of pola.
aSSB70A, -34A, and -7OB each inhibited SV40 DNA repli- The cvSSB monoclonal antibodies described in this report
cation by more than 70%, although pola has been thought to should continue to prove useful in the study of the molecular
be responsible only for lagging strand synthesis. This may be biology and biochemical functions of human SSB.
consistent with another function of SSB, or alternatively,
pola and SSB may be involved in the initiation of leading Acknowledgments-We thank Nilda Belgado and Barbara Phillips
strand synthesis. In fact, it has been shown recently that ~016 for technical assistance. We appreciate the help of Drs. Monika Lusky
does not efficiently utilize RNA primers, suggesting that polol and Lilian Clark with the immunoblotting experiments. We also
may be required for the initial stages of leading strand syn- thank Suk-Hee Lee, Takashi Matsumoto, Peter Bullock, and Toshih-
iko Eki for providing some of the enzymes used in this work. We are
thesis (Lee et al., 1989a). grateful to Drs. Frank Dean and Jim Borowiec for helpful comments
Although &SB70A, -34A, and -7OB blocked the stimula- on this manuscript.
tion of pola, aSSB70C inhibited DNA unwinding and ~016
stimulation. This is consistent with the observation that REFERENCES
various viral and prokaryotic SSBs stimulate ~016 and func- Borowiec, J. A., and Hurwitz, J. (1988a) Proc. N&l. Acad. Sci. U. 5’.
tion in the unwinding reaction, but the stimulation of pola is A. 85,64-68
specific to human SSB (Kenny et al., 1989). Thus, the stim- Borowiec, J. A., and Hurwitz, J. (198813) EMBO J. 7, 3149-3158
7700 Role of Human SSB in SV40 DNA Replication
Challberg, M. D., and Kelly, T. J. (1989) Annu. Reu. Biochem. 58, Natl. Acad. Sci. U. S. A. 86, 4877-4881
671-717 Mastrangelo, I. A., Hough, P. V. C., Wall, J. S., Dodson, M., Dean,
Chase, J. W., and Williams, K. R. (1986) Annu. Reu. Biochem. 55, F. B., and Hurwitz, J. (1989) Nature 338, 658-662
103-136 McEntee, K., Weinstock, G. M., and Lehman, I. R. (1980) Proc. Natl.
Dean, F. B., Borowiec, J. A., Ishimi, Y., Deb, S., Tegtmeyer, P., and Acad. Sci. U. S. A. 77.857-861
Hurwitz, J. (1987a) Proc. Nat!. Acad. Sci. 7% S. A. 84, 8267-8271 Murakami, Y., Wobbe, Cf. R., Weissbach, L., Dean, F. B., and Hurwitz,
Dean, F. B., Bullock, P., Murakami, Y., Wobbe, C. R., Weissbach, L., J. (1986) Proc. Natl. Acad. Sci. U. S. A. 83. 2869-2873
and Hurwitz, J. (1987b) Proc. Natl. Acad. Sci. U. S. A. 84, 16-20 O’Reilly, D. R., and Miller, L. K. (1988) J. Vbol. 62, 3109-3119
Dean, F. B., Dodson, M., Echols, H., and Hurwitz, J. (1987c) Proc. Prelich, G., and Stillman, B. (1988) Cell 53, 117-126
Natl. Acad. Sci. U. S. A. 84, 8981-8985 Prelich, G., Kostura, M., Marshak, D. R., Matthews, M. B., and
Dean, F. B., Lee, S.-H., Kwong, A. D., Bullock, P., Borowiec, J. A., Stillman, B. (1987) Nature 326,471-475
Kenny, M. K., Seo, Y. S., Eki, T., Matsumoto, T., Hodgins, G., and Roberts. J. M. (1989) Proc. N&l. Acad. Sci. U. S. A. 86, 3939-3943
Hurwitz. J. (1989) UCLA SvmD. Mol. Cell Biol. 127. 305-316 Siegel, L. M., and Monty, K. J. (1966) Biochim. Biop&s. Acta 112,
Deb, S. P.,‘and Tegtmeyer, P: (1’987) J. Viral. 61, 3649-3654 346-362
Dodson, M., Dean, F. B., Bullock, P., Echols, H., and Hurwitz., J. Stahl, H., Droge, P., and Knippers, R. (1986) EMBO J. 5,1939-1944
(1987) Science 238,964-967 Stillman, B. (1989) Ann. Rev. Cell Biol. 5, 197-245
Tiian. R. (1978) Cell 13. 165-179
Downey, K. M., Tan, C.-K., Andrews, D. M.. Li, X., and So, A. G.
Tsurimoto, T., and Stillman, B. (1989) Mol. Cell. Biol. 9, 609-619
(1988) Center Cells (Cold Spring Harbor) 6, 403-410
Williams. K. R.. and Chase. J. W. (1989) in The Biologv of Nonwecific
Fairman. M. P.. and Stillman. B. (1988) EMBO J. 7. 1211-1218
DNA-Ihoteii Interactioks (A. Revzin, ed) CRC Press, Inc., Boca
Hay, R. T., and’Russel1, W. Cl (1989) &o&em. J. 258, 3-16
Raton, FL, in press
Hager, D. A., and Burgess, R. R. (1980) Anal. Biochem. 109, 76-86 Wobbe, C. R., Dean, F., Weissbach, K., and Hurwitz, J. (1985) Proc.
Hsu, S.-M., Raine, L., and Fanger, H. (1981) J. Histochem. Cytochem.
Natl. Acad. Sci. U. S. A. 82, 5710-5714
29,577-580 Wobbe, C. R., Weissbach, L., Borowiec, J. A., Dean, F. B., Murakami,
Ikeda, J.-E., Enomoto, T., and Hurwitz, J. (1981) Proc. Natl. Acad. Y., Bullock, P., and Hurwitz, J. (1987) Proc. N&l. Aced. Sci. U. S.
Sci. U. S. A. 78,884-888 A. 84,1834-1838

Downloaded from http://www.jbc.org/ by guest on December 4, 2020

Ishimi, Y., Claude, A., Bullock, P., and Hurwitz, J. (1988) J. Biol. Wold, M. S., and Kelly, T. (1988) Proc. Natl. Acad. Sci. U. S. A. 85,
&em. 263, 19723-19733 2523-2527
Kenny, M. K., Lee, S.-H., and Hurwitz, J. (1989) Proc. Natl. Acad. Wold, M. S., Li, J. J., and Kelly, T. J. (1987) hoc. Natl. Acad. Sci.
Sci. U. S. A. 86,9757-9761 U. S. A. 84,3643-3647
Laemmli, U. K. (1970) Nature 227,680-685 Wold, M. S., Weinberg, D. H., Virshup, D. M., Li, J. J., and Kelly, T.
Lee, S.-H., Eki, T., and Hurwitz, J. (1989a) hoc. Natl. Acad. Sci. U. J. (1989) J. Biol. Chem. 264,2801-2809
S. A. 86, 7361-7365 Yang, L., Wold, M. S., Li, J. J., Kelly, T. J., and Liu, L. F. (1987)
Lee, S.-H., Kwong, A. D., Ishimi, Y., and Hurwitz, J. (1989b) hoc. Proc. Natl. Acad. Sci. U. S. A. 84, 950-954
 The role of human single-stranded DNA binding protein and its individual
subunits in simian virus 40 DNA replication.
M K Kenny, U Schlegel, H Furneaux and J Hurwitz
J. Biol. Chem. 1990, 265:7693-7700.

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