Human Topoisomerase I Promotes Initiation of Simian Virus 40 DNA Replication In Vitro

Abstract

Addition of purified human topoisomerase I (topo I) to simian virus 40 T antigen-driven in vitro DNA replication reactions performed with topo I-deficient extracts results in a greater than 10-fold stimulation of completed molecules as well as a more than 3-fold enhancement of overall DNA replication. To further characterize this stimulation, we first demonstrate that bovine topo I but not Escherichia coli topo I can also enhance DNA replication. By using several human topo I mutants, we show that a catalytically active form of topo I is required. To delineate whether topo I influences the initiation or the elongation step of replication, we performed delayed pulse, pulse-chase, and delayed pulse-chase experiments. The results illustrate that topo I cannot promote the completion of partially replicated molecules but is needed from the beginning of the reaction to initiate replication. Competitive inhibition experiments with the topo I binding T antigen fragment 1-246T and a catalytically inactive topo I mutant suggest that part of topo I’s stimulation of replication is mediated through a direct interaction with T antigen. Collectively, our data indicate that topo I enhances the synthesis of fully replicated DNA molecules by forming essential interactions with T antigen and stimulating initiation.

ACKNOWLEDGMENTS

This work was supported by PHS grant CA36118 to D.T.S.

We thank Pat Hearing for the 293 cells, Lance Stewart and Jim Champoux for the baculovirus expressing full-length human topo I and for purified Y723F, topo58, and topo12, Ken Marians for purified E. coli topo I, and Tom Melendy for purified bovine topo I.