The purified human single‐stranded DNA binding protein, replication protein A (RP‐A), forms specific complexes with purified SV40 large T antigen and with purified DNA polymerase alpha‐primase, as shown by ELISA and a modified immunoblotting technique. RP‐A associated efficiently with the isolated primase, as well as with intact polymerase alpha‐primase. The 70 kDa subunit of RP‐A was sufficient for association with polymerase alpha‐primase. Purified SV40 large T antigen bound to intact RP‐A and to polymerase‐primase, but not to any of the separated subunits of RP‐A or to the isolated primase. These results suggest that the specific protein‐protein interactions between RP‐A, polymerase‐primase and T antigen may play a role in the initiating of SV40 DNA replication.

The single-stranded DNA binding protein RP-A is required in SV40 DNA in vitro replication. The RP-A purified from calf thymus contains 4 polypeptides with molecular weights 70kDa, 53kDa, 32kDa, and 14kDa. The p70 subunit and its proteolysed form p53 are recognized by the monoclonal antibody 70C (Kenny et al. (1990)) and bind to ssDNA. The p70 and p32 subunits of bovine RP-A are phosphorylated by CDC2-cyclin B kinase. Bovine RP-A supports the origin dependent unwinding of SV40 DNA by T antigen. Furthermore, bovine RP-A can efficiently substitute for human RP-A in SV40 DNA replication in vitro. A modified blotting technique revealed that RP-A interacts specifically and directly with the p48 subunit of DNA polymerase alpha-primase complex.

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