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,J Hurwitz

doi:10.1016/s0021-9258(19)39170-7

Abstract

Human single-stranded DNA binding protein (human SSB) is a multisubunit protein containing polypeptides of 70, 34, and 11 kDa that is required for SV40 DNA replication in vitro. In this report we identify 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 alpha SSB70A, alpha SSB70B, alpha SSB70C, and alpha SSB34A 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) alpha and delta. The DNA unwinding reaction and stimulation of pol delta were blocked by alpha SSB70C, whereas the stimulation of pol alpha by human SSB was unaffected by this antibody. Conversely, alpha SSB70A, -70B, and -34A inhibited the stimulation of pol alpha, but they had no effect on DNA unwinding and pol delta stimulation. None of the antibodies inhibited the binding of SSB to single-stranded DNA. These results suggest that DNA unwinding and stimulation of pol alpha and pol delta are required functions of human SSB in SV40 DNA replication. The human SSB 70-kDa subunit appears to be required for DNA unwinding and pol delta stimulation, whereas both the 70- and 34-kDa subunits may be involved in the stimulation of pol alpha.

Highlights

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 effect of &SB monoclonal antibodies (Ab) on reactions involving 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 ulatory effects of human SSB on pola and ~016 are likely to occur by different mechanisms

Summary

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 BiotechnologyInc.); radionucleotides (Du Pont-New England Nuclear); unlabeled nucleotides, M13mp DNA, Ml3 sequencing primer (dTCCCAG-TCACGACGT), DNase I, and E. coli DNA polymerase I (BoehringerMannheim); creatine phosphokinase (Worthington); chloroquine (Sterling Drug Inc.); protein A-Sepharose (Sigma); goat anti-mouse agarose (HyClone); immunoblotting reagents (Bio-Rad); immunohistochemical reagents (Vector Laboratories).Bovine serum albumin (BSA, Miles Laboratories) was heat-denatured before use. Reagents-The following reagents were obtained commercially: p~ly(dA)~~~ (Life Sciences); micrococcal nuclease, E. coli SSB, T4g32, Sephadex G-25, and ohgo(dT),. Inc.); radionucleotides (Du Pont-New England Nuclear); unlabeled nucleotides, M13mp DNA, Ml3 sequencing primer TCACGACGT), DNase I, and E. coli DNA polymerase I Mannheim); creatine phosphokinase (Worthington); chloroquine (Sterling Drug Inc.); protein A-Sepharose (Sigma); goat anti-mouse agarose (HyClone); immunoblotting reagents (Bio-Rad); immunohistochemical reagents (Vector Laboratories). SV40 origin-containing plasmid pSVOlAEP has been described previously (Wobbe et al, 1985). Crude extract (Wobbe et aZ., 1985), topo I (Ishimi et al, 1988), pol~lprimase (Ishimi et al, 1988), PCNA-dependent. ~016 (Lee et al, 1989b), Role of Human SSB in SV40 DNA Replication. PCNA (Lee et a!., 1988), and the 0.4 M double-stranded

Results

Discussion

Conclusion