The proposed studies represent a continuation of our long term goals of understanding the enzymology and functions of human DNA polymerase delta, a central enzyme in mammalian chromosomal DNA replication. The broad themes of the proposal are 1) the characterization of the DNA polymerase delta heterotetramer and its subassemblies, and the identification of novel accessory proteins with the goal of understanding the architecture of the mammalian replication complex, 2) the characterization of the interactions between pol delta and PCNA, and 3) the investigation of the molecular linkages between regulatory systems that control cellular DNA replication and pol delta. These studies build on previous and current ongoing work in our laboratory during which time we have developed the tools and necessary expertise. We will use as a platform for our studies the reconstituted pol delta heterotetramer which includes the two new subunits that we have discovered. We will investigate the molecular organization of the subunits, and the physical and functional interactions of the complex with PCNA. The contribution of individual subunits of pol delta to the binding of PCNA will be determined. A high molecular weight form of pol delta will be further purified to identify the protein(s) that associate with the pol delta heterotetramer. Two novel proteins, p38 and p46, that interact with the p50 subunit, will be studied as putative accessory proteins/subunits of pol delta. The interaction of p38 and p46 with pol delta will be analyzed by determining if they can be reconstituted into pol delta complexes. Their potential functions will be investigated by a combination of biochemical, molecular biological and cell biological approaches. The cell cycle dependence of the phosphorylation of the subunits of the pol delta enzyme in vivo will be investigated, and the role of the cyclin/cdks as the mediator kinases will be established. Tryptic peptide mapping of labeled subunits will be used to identify those sites whose phosphorylation levels are a) cell cycle dependent and b) inhibited by cyclin/cdk inhibitors. We will investigate phosphorylation of pol delta by the protein kinases involved in cell cycle checkpoint signaling. Functional consequences of phosphorylation of pol delta will be investigated. These studies are directed to a) establishing if p125 is a cellular target of these kinases, investigation of the in vivo phosphorylation of p125 during DNA damage, and examination the functional consequences of phosphorylation of pol delta. The role of p68 as a targeting protein for the recruitment of protein phosphatase-1 to the pol delta complex will be investigated.
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