Topoisomerase I stimulates SV40 T antigen-mediated DNA replication and inhibits T antigen's ability to unwind DNA at nonorigin sites.
We have previously found that purified SV40 T antigen and topoisomerase I (topo I) bind to one another in vitro. In this report, we determined the effects of human topo I on T antigen-mediated DNA replication and investigated whether it altered T antigen's biochemical activities. Topo I stimulates DNA replication and especially increases the amounts of finished circular molecules. This protein had no effect on T antigen's ability to bind, distort, or unwind the origin of replication. However, unwinding of DNA by T antigen was strongly inhibited by topo I when it was initiated at sites other than the origin. We demonstrate that the presence of T antigen binding sites in DNA interfere with inhibition of unwinding by topo I. These results indicate that topo I may increase the specificity of unwinding by inhibiting the reaction at non-origin sites. Fragments of T antigen that bind to topo I abrogate topo I's inhibition of non-origin-dependent unwinding, indicating that topo I inhibits unwinding through a direct interaction with T antigen. We propose a model whereby T antigen and topo I function together at the origin to specifically unwind it and initiate DNA replication.
Simmons
D T
DT
Department of Biological Sciences, University of Delaware, Newark 19716-2590, USA. dsimmons@udel.edu
Trowbridge
P W
PW
Roy
R
R
D000952:Antigens, Polyomavirus Transforming / Q000187:drug effects / Q000378:metabolism*; D004261:DNA Replication / Q000187:drug effects*; D004264:DNA Topoisomerases, Type I / Q000502:physiology*; D004279:DNA, Viral / Q000187:drug effects / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D018741:Replication Origin / Q000187:drug effects / Q000502:physiology
D000952:Antigens, Polyomavirus Transforming; D004279:DNA, Viral; D004264:DNA Topoisomerases, Type I