TOPOISOMERASE-I STIMULATES SV40 T-ANTIGEN-MEDIATED DNA-REPLICATION AND INHIBITS T-ANTIGENS ABILITY TO UNWIND DNA AT NONORIGIN SITES

We have previously found that purified SV40 T antigen and topoisomeias e I (topo I) bind to one another in vitro. in this report, we determin ed the effects of human topo I on T antigen-mediated DNA replication a nd investigated whether it altered T antigen's biochemical activities. Topo I stimulates DNA replication and especially increases the amount s of finished circular molecules. This protein had no effect on T anti gen's ability to bind, distort, or unwind the origin of replication. H owever, 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 inh ibition of unwinding by topo I. These results indicate that topo I may increase the specificity of unwinding by inhibiting the reaction at n on-origin sites. Fragments of T antigen that bind to topo I abrogate t opo 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. (C ) 1998 Academic Press.