Simmons D T, Trowbridge P W, Roy R
Department of Biological Sciences, University of Delaware, Newark 19716-2590, USA.
Virology. 1998 Mar 15;242(2):435-43. doi: 10.1006/viro.1997.9024.
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.
我们之前发现，纯化的SV40 T抗原和拓扑异构酶I（拓扑酶I）在体外会相互结合。在本报告中，我们确定了人拓扑酶I对T抗原介导的DNA复制的影响，并研究了它是否改变了T抗原的生化活性。拓扑酶I刺激DNA复制，尤其增加了完整环状分子的数量。这种蛋白质对T抗原结合、扭曲或解开复制起点的能力没有影响。然而，当T抗原在复制起点以外的位点启动DNA解旋时，拓扑酶I会强烈抑制这种解旋。我们证明，DNA中T抗原结合位点的存在会干扰拓扑酶I对解旋的抑制作用。这些结果表明，拓扑酶I可能通过抑制非起点位点的反应来提高解旋的特异性。与拓扑酶I结合的T抗原片段消除了拓扑酶I对非起点依赖性解旋的抑制作用，这表明拓扑酶I通过与T抗原的直接相互作用来抑制解旋。我们提出了一个模型，即T抗原和拓扑酶I在起点共同发挥作用，特异性地解开起点并启动DNA复制。
