The overall goals of this project are to elucidate the structure, function and regulation of the mammalian replication complex and the mechanisms by which replication fidelity is maintained in mammalian cells. The major focus of the proposed studies is DNA polymerase d, an essential DNA polymerase which is required for replication of chromosomal DNA and may also be involved in DNA repair. The investigator's have recently succeeded in overexpressing the two subunits of DNA polymerase d (p125 and p50) in baculovirus-infected insect cells, purified and characterized the individual recombinant subunits as well as the recombinant heterodimer, and established that the small subunit is essential for functional interaction of DNA polymerase d with the proliferating cell nuclear antigen (PCNA) and thus for highly processive DNA synthesis. They propose to continue studies on the potential role of the small subunit of DNA polymerase d as a central organizer of the eukaryotic replisome, to elucidate the mechanisms regulating the activity of DNA polymerase d, particularly as a function of cell cycle position, to define the domains which interact in the heterodimer and with PCNA, to determine the residues in the catalytic subunit that are critical for fidelity of DNA synthesis and to determine whether residues which are altered in some sporadic colorectal cancers affect proofreading by DNA polymerase d. They also propose to pursue studies on another putative replication protein, d helicase, to determine its role, if any, in mammalian DNA replication.
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