Initiation of DNA replication is a complicated process that is understood in only very broad terms. At present, the best eukaryotic model for studies of initiation of DNA replication is the Simian Virus 40 (SV4O) in vitro replication system. Initiation of SV4O replication requires a single viral protein termed T-antigen (T-ag). Critical roles played by T.ag during initiation of replication include site specific binding to the SV4O origin, catalysis of subsequent unwinding events and recruitment of additional proteins necessary for initiation of DNA synthesis. However, our understanding of these events is limited owing to a lack of structural information about T-ag or the T-ag DNA binding domain (T-ag-bd). The following specific aims are proposed to address these issues. I. To determine the structure of the DNA binding domain of SV4O T-antigen. II. To characterize the biochemical properties of the T-ag-bd, and of certain mutant forms of the T-ag-bd, and to examine the structures of those mutant forms of the T-ag-bd with interesting properties. III. To delineate the interaction of the T-ag-bd with DNA. The work proposed in this application is significant from a basic science standpoint because it will provide the first structure of a protein domain that recognizes an origin of replication, whether from a prokaryotic or eukaryotic source. Moreover, existing sequence data indicate the T-ag-bd is not related to other known DNA binding proteins, suggesting the structure may reveal a new protein structural motif. A knowledge of this structure, or of the structure of this domain complexed to DNA, will contribute significantly to advancing our understanding of replication by revealing the protein/DNA contacts that enable specific recognition and binding to an origin of replication. The health related significance of the work proposed stems from the fact that SV4O T-ag is very homologous to the T-ags encoded by the BK and JC viruses. These viruses induce a number of diseases in humans, including cancer. For example, JC virus induces progressive multifocal leukoencephalopathy, a disease present in many AIDS patients.
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