Simmons Daniel T, Gai Dahai, Parsons Rebekah, Debes Amanda, Roy Rupa
Department of Biological Sciences, University of Delaware, Newark, DE 19716-2590, USA.
Nucleic Acids Res. 2004 Feb 11;32(3):1103-12. doi: 10.1093/nar/gkh236. Print 2004.
The assembly of the complex that forms over the simian virus 40 origin to initiate DNA replication is not well understood. This complex is composed of the virus-coded T antigen and three cellular proteins, replication protein A (RPA), DNA polymerase alpha/primase (pol/prim) and topoisomerase I (topo I) in association with the origin. The order in which these various proteins bind to the DNA was investigated by performing binding assays using biotinylated origin DNA. We demonstrate that in the presence of all four proteins, pol/prim was essential to stabilize the initiation complex from the disruptive effects of topo I. At the optimal concentration of pol/prim, topo I and RPA bound efficiently to the complex, although pol/prim itself was not detected in significant amounts. At higher concentrations less topo I was recruited, suggesting that DNA polymerase is an important modulator of the binding of topo I. Topo I, in turn, appeared to be involved in recruiting RPA. RPA, in contrast, seemed to have little or no effect on the recruitment of the other proteins to the origin. These and other data suggested that pol/prim is the first cellular protein to interact with the double-hexameric T antigen bound to the origin. This is likely followed by topo I and then RPA, or perhaps by a complex of topo I and RPA. Stoichiometric analysis of the topo I and T antigen present in the complex suggested that two molecules of topo I are recruited per double hexamer. Finally, we demonstrate that DNA has a role in recruiting topo I to the origin.
在猿猴病毒40复制起点上形成以启动DNA复制的复合物的组装过程尚未完全清楚。该复合物由病毒编码的T抗原和三种细胞蛋白组成，即复制蛋白A（RPA）、DNA聚合酶α/引发酶（pol/prim）和拓扑异构酶I（topo I），它们与复制起点结合。通过使用生物素化的复制起点DNA进行结合试验，研究了这些不同蛋白质与DNA结合的顺序。我们证明，在所有四种蛋白质存在的情况下，pol/prim对于稳定起始复合物免受topo I的破坏作用至关重要。在pol/prim的最佳浓度下，topo I和RPA能有效地与复合物结合，尽管未检测到大量的pol/prim本身。在较高浓度下，招募的topo I较少，这表明DNA聚合酶是topo I结合的重要调节剂。反过来，topo I似乎参与招募RPA。相比之下，RPA对其他蛋白质招募到复制起点的影响似乎很小或没有影响。这些以及其他数据表明，pol/prim是第一个与结合在复制起点上的双六聚体T抗原相互作用的细胞蛋白。随后可能是topo I，然后是RPA，或者可能是topo I和RPA的复合物。对复合物中存在的topo I和T抗原进行化学计量分析表明，每个双六聚体招募两个topo I分子。最后，我们证明DNA在将topo I招募到复制起点方面发挥作用。
