Structure and Function of SV40 Large Tumor Antigen

描述(申请人提供)：SV40DNA的复制已经被许多研究人员作为真核生物，更具体地说是哺乳动物DNA复制的范例而研究。尽管参与SV40DNA合成的分子已知，其许多功能也已阐明，但我们对DNA复制机制的了解仍存在严重差距。特别是，我们对SV40 DNA上起始和延伸复合体的组装方式以及有效合成DNA所需的蛋白质-蛋白质和蛋白质-DNA相互作用知之甚少。对于DNA复制过程中各种事件的时间安排，人们只有初步的了解。因此，我们的知识状态只允许对DNA合成的机制有一个粗略的了解。为了获得这些信息，我们制定了三个具体目标。 1.研究SV40大T抗原在原点组装成功能性双六聚体的条件。这一目标将主要通过研究在组装中有缺陷的T抗原突变来实现。 2.阐明T抗原起始机解开起始点的机制和事件发生的时间。将采取各种方法来推断这是如何运作的。我们的目标是推断在解开过程中DNA和T抗原发生的变化 3.对起始和延伸复合体的组装进行表征，以便更好地了解DNA复制的细节。我们将研究复合体组装的顺序，以及各种组件如何共同进行DNA复制。 预计对SV40DNA复制机制的更好理解将延伸到哺乳动物DNA复制的特征，并提供针对复制因子和开发足够的治疗方法来治疗人类疾病(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.