The broad, long-term objectives of this project are; 1) to identify and characterize enzymatic functions involved in human DNA repair, 2) to utilize this information to elucidate the various pathways for DNA repair in human cells and 3) to establish specific connections between defective DNA repair and a predisposition to cancer. Substantial evidence has accrued indicating that cancer is the result of a series of mutations in genes that affect the growth and replication of a cell (oncogenes and/or genes that suppress uncontrolled growth, the tumor suppressor genes). It is now a well established fact that cells have a complex set of enzymatic DNA repair mechanisms which protect the genome against mutation. It is conceivable that, within the heterogeneous human population, cells from different individuals differ in their abilities to repair DNA. If this repair is defective or compromised, an increased mutation rate would result, which in turn would increase the likelihood of mutation in oncogenes or tumor suppressor genes. In a diagnostic sense, an understanding of the mechanisms of human DNA repair would serve to warn individuals, with ineffective repair, that they may predisposed to cancer. These individuals could be monitored regularly for early diagnosis of cancer and also removed from environmental situations augmenting mutation and carcinogenesis.
The specific aims of this project are; 1) to characterize the human DNA repair function, uracil-DNA glycosylase, from normal cells and cells derived from individuals with genetic disorders showing a predisposition to cancer, 2) to characterize the gene encoding human dUTP nucleotidohydrolase (dUTPase) in normal and genetically aberrant cells, 3) to determine the significance of post-translational phosphorylation of the human dUTPase protein, 4) to analyze DNA repair functions of the herpes simplex virus. Utilizing cloning methodologies, Southern blot analysis and DNA sequencing, the structural characteristics of the glycosylase and dUTPase genes will be determined. Cell culture, northern blot and western blot analysis will be used to examine expression of these genes in normal and genetically abnormal cells. Cell culture, biochemical and immunochemical techniques will be used to examine the post translational phosphorylation of human dUTPase as a function of the proliferative state of the cell. Similar molecular, immunological and biochemical methodologies will be used to characterize the herpes simplex virus DNA repair functions.
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