The overall objective of this research is to provide quantitative information on the yield, variety and fate of ionizing radiation- induced DNA base damage in cells and in DNA models in vitro. Cell models are selected from strains of Escherichia coli and related procaryotes that have genetically characterized mutations in genes specific for various DNA repair enzymes. Selected Strains include cells deficient in excision repair (e.g., uvrABC mutants); inducible repair (e.g., lexA, recA); exonucleosidic processing (recBC., xth, etc.) and other pertinent repair pathways. In this phase of our long range research, we will add studies on radiation- induced base damage in cells that are deficient in catalase (katEFG) and/or superoxide dismutase enzymes (sodAB). These studies will aid in determining the effects of the molecular radiolysis products, O2- and H2O2, on cellular DNA damage without exogenous chemical modifiers such as H2O2 or paraquat. Procedures for the measurement of DNA base damage in cells and in model DNA that have been developed in previous years include chromatography, radiolabeling, and monoclonal antibody assays, chromatography procedures include multiproduct HPLC assays of DNA nucleoside or base hydrolysates using both UV and radioactive flow detection. Monoclonal antibody assays include recently-developed ELISA procedures to measure thymine glycol and dihydrothymine in cellular DNA isolates. These procedures will be used in (ongoing) studies on thymine base damage and in future studies on purine and cytosine damage. Since most of the initial yield of chemical DNA damage is cells is formed and the yields by cellular repair or exogenous chemicals. Research providing insight into these mechanisms will benefit our understanding of radiation cell killing and mutagenesis.
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