| Abstract: |
Simian virus 40 (SV40) large tumor antigen (T antigen) binds to two contiguous sites at the SV40 origin of replication. Of these two sites, I and II, only site II is critical for replication. We have studied the interaction between T antigen and these sites by two methods--nitrocellulose filter binding and DNase I protection. We show that T antigen binds with high occupancy to site I at 0 degrees C, 25 degrees C, and 37 degrees C but to site II only at 0 degrees C and 25 degrees C. At 37 degrees C, the temperature essential for the initiation of SV40 DNA replication in vitro, ATP is required for the interaction of T antigen and site II. ATP can be replaced efficiently by adenosine 5'-[beta,gamma-imido]triphosphate and ADP, suggesting that hydrolysis of the nucleotide is not essential for the binding of T antigen to site II. The binding to the region critical for replication can occur in the presence of a variety of nucleoside triphosphates; dATP supports binding at a concentration 1/30th that of ATP, while dGTP and rGTP were inactive at all concentrations tested. |
