THE REPLICATION PROTEIN-A BINDING-SITE IN SIMIAN-VIRUS-40 (SV40) T-ANTIGEN AND ITS ROLE IN THE INITIAL STEPS OF SV40 DNA-REPLICATION

Physical interactions of simian virus 40 (SV40) large tumor (T) antige n with cellular DNA polymerase alpha-primase (PoI/Prim) and replicatio n protein A (RPA) appear to be responsible for multiple functional int eractions among these proteins that are required for initiation of vir al DNA replication at the origin, as well as during lagging-strand syn thesis. In this study, we mapped an RPA binding site in T antigen (res idues 164 to 249) that is embedded within the DNA binding domain of T antigen. Two monoclonal antibodies whose epitopes map within this regi on specifically interfered with RPA binding to T antigen but did not a ffect T-antigen binding to origin DNA or Pol/Prim, ATPase, or DNA heli case activity and had only a modest effect on origin DNA unwinding, su ggesting that they could be used to test the functional importance of this RPA binding site in the initiation of viral DNA replication. To r ule out a possible effect of these antibodies on origin DNA unwinding, we used a two-step initiation reaction in which an underwound templat e was first generated in the absence of primer synthesis. In the secon d step, primer synthesis was monitored with or without the antibodies. Alternatively, an underwound primed template was formed in the first step, and primer elongation was tested,vith or without antibodies in t he second step. The results show that the antibodies specifically inhi bited both primer synthesis and primer elongation, demonstrating that this RPA binding site in T antigen plays an essential role in both eve nts.