Our long-term goal is a molecular definition of the repair of oxidative DNA damages in eukaryotic cells. This effort is being driven by the recognition that reactive oxygen species are widespread in aerobic biology, being produced as by-products of oxygen metabolism and also by numerous environmental agents. Since it is also becoming clear that cellular defenses against oxidative damage act to limit even """"""""spontaneous"""""""" mutagenesis, a biochemical understanding of oxidized DNA repair will shed light on a fundamental process in genetic toxicology. The thrust of the present application is based in our prior biochemical analysis of a major pathway for repair of free-radical damages in the simple eukaryote Saccharomyces cerevisiae, as embodied in the major 3'-repair diesterase/AP endonuclease (Ape) of yeast. This activity initiates excision repair by removing 3'-fragments that block DNA synthesis, as well as incising at base-free sites that can block synthesis and act as mutation targets. The cloned APE gene will be used to address critically the role played by the Ape protein in cellular antioxidant defense. The proposed work falls into four main areas that address related questions: (i) manipulation of APE and SOD1 gene expression primarily in yeast, but also in bacteria and HeLa cells; (ii) the effect of Ape and Sod1 expression on cell killing and mutagenesis by both intrinsic and exogenous oxidants; (iii) correlation of specific oxidative damages with mutagenesis and cell killing; (iv) detection of additional control or repair pathways that operate in conjunction with Ape. The expression studies will employ gene disruption, and insertion of APE into different regulated expression systems that work in yeast, and in E. coli and HeLa cells. The resulting yeast strains will be tested both for spontaneous aerobic mutagenesis and killing, and for that induced by redox mutagens. For bacteria and HeLa cells, the analysis will be restricted to cell killing studies. Sensitive and specific biochemical assays will be used to quantitate particular DNA damages in enzyme-deficient yeast, and the data correlated with the results of the mutation studies in an effort to reveal whether the natural Ape substrates can be mutagenic if left unrepaired. Both a genetic approach (mutant isolation) and a biochemical approach (establishment of a specific assay) will be exploited to determine what other systems act in the antimutagenic repair of oxidative DnA damages, focusing particularly on the lesion 8- hydroxyguanine. These studies will establish detailed characteristics of the role of the yeast APE gene product in preventing the genotoxicity of oxidative agents, and provide an initial description of additional pathways that act in concert with Ape protein. The work will yield the most biochemically detailed picture yet obtained of a key pathway in redox repair in eukaryotes.
Showing the most recent 10 out of 77 publications
