Structure and Function of SV40 Large Tumor Antigen

描述（由申请人提供）：许多研究者已经研究了SV 40 DNA的复制，将其作为真核生物和更具体地哺乳动物DNA复制的范例。虽然参与SV 40 DNA合成的分子是已知的，并且它们的许多功能已经阐明，但我们对DNA复制机制的认识存在严重的空白。特别是，我们对起始和延伸复合物在SV 40 DNA上组装的方式以及有效DNA合成所需的蛋白质-蛋白质和蛋白质-DNA相互作用知之甚少。对DNA复制过程中各种事件的时间只有初步的了解。因此，我们的知识水平只允许对DNA合成的机制进行粗略的描述。为了获得这些信息，我们制定了三个具体目标。 1.研究SV 40大T抗原在起点组装成功能性双六聚体的要求。这一目标将主要通过研究在装配中有缺陷的T抗原突变体来实现。 2.阐明T抗原起始机器解旋起点时发生的事件的时间和解旋起点的机制。将采取各种方法来推断这是如何工作的。 我们的目标是推断DNA和T抗原在解旋过程中发生的变化 3.为了表征起始和延伸复合物的组装，以便更好地了解DNA复制的细节。我们将研究复合物组装的顺序，以及各种成分如何协同工作以进行DNA复制。 预计对SV 40 DNA复制机制的更好理解将扩展到哺乳动物DNA复制的表征，并提供靶向复制因子和开发适当疗法以治疗人类疾病（如癌症）所需的基本知识。

DESCRIPTION (provided by applicant): The replication of SV40 DNA has been studied by many investigators as a paradigm for the replication of eukaryotic and more specifically mammalian DNA. Although the molecules involved in the synthesis of SV40 DNA are known and many of their functions elucidated, there are serious gaps in our knowledge of the mechanism of DNA replication. In particular, we understand little of the way in which initiation and elongation complexes assemble on SV40 DNA and of the protein-protein and protein-DNA interactions that are needed for efficient DNA synthesis. There is only rudimentary understanding of the timing of various events during DNA replication. Our state of knowledge therefore allows only a sketchy picture of the mechanics of DNA synthesis. To obtain this information, we have developed 3 specific aims. 1. To study the requirements for assembly of SV40 large T antigen into functional double hexamers on the origin. This aim will be carried out primarily by studying mutants of T antigen that are defective in assembly. 2. To elucidate the mechanism of origin unwinding and timing of events that occur when the origin is unwound by the T antigen initiation machine. Various approaches will be taken to deduce how this works. Our goal here is to deduce the changes that take place to the DNA and to T antigen during unwinding 3. To characterize the assembly of initiation and elongation complexes so as to better understand the details of DNA replication. We will study the order in which complexes assemble and how the various components work together to carry out DNA replication. It is anticipated that a much better understanding of the mechanism of SV40 DNA replication will spill over to the characterization of mammalian DNA replication and provide the basic knowledge that is required to target replication factors and develop adequate therapies to treat human diseases such as cancer.