STRUCTURE/FUNCTION OF THE SV40 LARGE TUMOR ANTIGEN

虽然我们已经了解了大量的蛋白质参与 真核DNA复制，我们对它们如何复制知之甚少。 它们相互沟通，启动DNA合成， 延伸反应。几乎我们所知道的关于这个过程的一切， 高等真核生物是从对SV 40的研究中产生的。这种病毒编码为 只有一种蛋白质直接参与DNA合成;其余的 这些因素来自细胞。T抗原与许多这些 蛋白质，包括DNA聚合酶α、RPA和拓扑异构酶I （topoI），并且有越来越多的证据表明，T 抗原与这些细胞蛋白是复制所需的， SV40 DNA我们集中讨论了T抗原与 topoI，并已获得证据表明，这两种蛋白质可能形成 复制起点的第一个功能复合体。我们的目标是 在这些观察的基础上， 这些细胞蛋白质相互结合形成 起始复合物，并确定每种蛋白质的功能， 引发和延伸。特别是，我们希望更好地 了解T抗原解旋酶与 拓扑异构酶来更好地理解这两种酶是如何交流的 在DNA解旋过程中相互作用。由于这个综合体必须处理 在解旋过程中产生的单链， 我想表征T抗原的单链DNA结合结构域 我们最近发现它与原点完全分离 结合域因此，我们想分析拓扑异构酶I的作用 在SV 40 DNA复制中，研究复合物的组成， 参与SV 40 DNA复制，并决定每一个基因的功能。 蛋白质在复合物中，并表征单一的功能， T抗原单链DNA结合结构域 解旋酶的作用机制

Although we have learned a great deal about the proteins involved in eukaryotic DNA replication, we know relatively little about how they communicate with one another to initiate DNA synthesis and to carry out the elongation reaction. Nearly everything we know about this process in high eukaryotes has come from the study of SV40. This virus codes for only one protein directly involved in DNA synthesis; the remaining factors come from the cell. T antigen associates with a number of these proteins, including DNA polymerase alpha, RPA and topoisomerase I (topoI) and there is accumulating evidence that the interaction of T antigen with these cellular proteins is required for the replication of SV40 DNA. We have concentrated on the association between T antigen and topoI and have obtained evidence that these two proteins may form the first functional complex at the origin of replication. Our goal is to build on these observations to obtain a clearer picture of the order in which cellular proteins associated with one another to form the initiation complex and to determine the function of each protein during initiation and elongation. In particular, we would like to better understand the relationship between the T antigen helicase and topoisomerase to better appreciate how these two enzymes communicate with one another during DNA unwinding. Since this complex must deal with the single strands that are generated during unwinding, we would also like to characterize the single-stranded DNA binding domain of T antigen which we have recently shown to be completely separate from the origin- binding domain. We therefore want to analyze the role of topoisomerase I in SV40 DNA replication, study the composition of the complex(es) that participates in SV40 DNA replication and determine the function of each protein in the complex, and characterize the function of the single- stranded DNA binding domain of T antigen in an effort to gain insights into the mechanism of helicase action.