The overall goal of this proposal is to continue work on the characterization of enzymes involved in nucleotide metabolism. Also included are initial studies that attempt to elucidate the mechanisms responsible for regulated fluctuation of certain enzyme activities with the DNA synthesis phase of the cell cycle. These studies may eventually aid in determining the efficacy of anticancer agents such as methotrexate, cytosine arabinoside and related analogs. Futhermore, uncovering basic information of how the cell regulates the synthesis of the machinery necessary for its own proliferation may lead to an understanding of growthcontrolling elements. The aberrant growth regulation of a cancer cell may then begin to be understood. This research proposal has three main aspects: 1. Further characterization of dUTPase, the enzyme responsible for dUTP hydrolysis in the cell. This will entail the use of monoclonal antibodies already generated against the dUTPase enzyme derived from HeLa S3 cells. An attempt will be made to elucidate the reason for post-translational phosphorylation of the dUTPase protein. 2. Generations of monoclonal antibodies against SDS polyacrylamide gel purified enzyme that apparently hydrolyzes dCTP within the cell. These antibodies will then be used to characterize this dCTPase enzyme in terms of its structure and function. 3. Analysis of thymidine kinase and dUTPase from human cells in terms of whether posttranslational modification occurs as a cell cycle dependent modification, coordinate with their enzyme activities. This aspect of the proposal will utilize methods of cell synchronization, immunoprecipitation and enzymatic assays for the analysis of the proteins under study.
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