The Replication Protein A Binding Site in Simian Virus 40 (SV40) T Antigen and Its Role in the Initial Steps of SV40 DNA Replication

Abstract

Physical interactions of simian virus 40 (SV40) large tumor (T) antigen with cellular DNA polymerase α-primase (Pol/Prim) and replication protein A (RPA) appear to be responsible for multiple functional interactions among these proteins that are required for initiation of viral DNA replication at the origin, as well as during lagging-strand synthesis. In this study, we mapped an RPA binding site in T antigen (residues 164 to 249) that is embedded within the DNA binding domain of T antigen. Two monoclonal antibodies whose epitopes map within this region specifically interfered with RPA binding to T antigen but did not affect T-antigen binding to origin DNA or Pol/Prim, ATPase, or DNA helicase activity and had only a modest effect on origin DNA unwinding, suggesting that they could be used to test the functional importance of this RPA binding site in the initiation of viral DNA replication. To rule out a possible effect of these antibodies on origin DNA unwinding, we used a two-step initiation reaction in which an underwound template was first generated in the absence of primer synthesis. In the second 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 with or without antibodies in the second step. The results show that the antibodies specifically inhibited both primer synthesis and primer elongation, demonstrating that this RPA binding site in T antigen plays an essential role in both events.

Structural mechanism of RPA loading on DNA during activation of a simple pre-replication complex

“…When purified monoclonal antibody against the extreme C-terminus of Tag was titrated into the binding reaction, the mobility of the ssDNA complex remained unchanged (lanes 4-6). Two other antibodies that recognize epitopes remote from the Tag-OBD and do not interfere with RPA:Tag complex formation (Weisshart et al, 1998) yielded identical results (not shown). These results indicated that Tag is not present in RPAII.…”

“…The viral replicative DNA helicase, SV40 T antigen (Tag), binds to RPA via its origin DNA-binding domain (OBD, residues 131-259) and this interaction is essential for SV40 DNA replication (Dornreiter et al, 1992;Melendy and Stillman, 1993;Weisshart et al, 1998). The Tag-binding regions of RPA have been mapped to RPA70 residues 169-327 (Braun et al, 1997) or RPA70A (Loo and Melendy, 2004;Park et al, 2005), and RPA32C (Lee and Kim, 1995).…”

Episomal vectors for gene expression in mammalian cells

“…These interactions are complex and much remains to be learned about the protein–DNA interactions that take place at the ori, the structural consequences of these interactions, and the way these processes are regulated. This has been studied in much more detail for SV40 [38–40]. Considering the high degree of homology between the two viruses, it is likely that a similar mechanism is used by BKV.…”

The Werner Syndrome: A Model for the Study of Human Aging

“…SV40 T antigen binds to RPA1 (p70) and to DNA polymerase alfa/ primase and in this way initiates replication of the viral genome. 78,79 RPA is required during the elongation step of DNA synthesis. 79,80 WRN shares a number of features with SV40 large T antigen, which is capable of extending life in vitro.…”

“…79,80 WRN shares a number of features with SV40 large T antigen, which is capable of extending life in vitro. 78,79…”