Structure and Function of SV40 Large Tumor Antigen

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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.