Topoisomerase I associates specifically with simian virus 40 large-T-antigen double hexamer-origin complexes

Topoisomerase I (topo I) is required for releasing torsional stress during simian virus 40 (SV40) DNA replication, Recently, it has been demonstrated that topo I participates in initiation of replication as well as in elongat ion. Although T antigen and topo I can bind to one another in vitro, there is no direct evidence that topo I is a component of the replication initiat ion complex, We demonstrate in this report that topo I associates with T-an tigen double hexamers bound to SV40 origin DNA (T-DH but not to single hexa mers. This association has the same nucleotide and DNA requirements as thos e for the formation of double hexamers on DNA. Interestingly, topo I prefer s to bind to fully formed T-DH complexes over other oligomerized forms of T antigen associated with the origin. High ratios of topo I to origin DNA de stabilize T-DH. The partial unwinding of a small-circular-DNA substrate is dependent on the presence of both T antigen and topo I but is inhibited at high topo I concentrations. Competition experiments with a topo I-binding f ragment of T antigen indicate that an interaction between T antigen and top o I occurs during the unwinding reaction, We propose that topo I is recruit ed to the initiation complex after the assembly of T-DH and before unwindin g to facilitate DNA replication.