Application): Mechanisms of action of oxidative DNA-damaging agents and cellular responses to these agents are problems of considerable medical importance. However, all of the mechanisms cells use to defend against and repair the damage are not known. The long-term goal of this project is to determine mechanisms cells use to protect against oxidative damage. Specifically, the long- term objectives are to understand DNA repair pathways, recovery processes, and proteins important in cellular responses to oxidative DNA-damaging agents. Three classes of oxidative DNA-damaging agents are included in the study: ionizing radiation, anti-cancer bleomycins and structurally related phleomycins, and hydrogen peroxide. The bleomycin group of antibiotics is an important therapeutic agent useful as a single agent in treating several human cancers and widely used in combination chemotherapy and radiotherapy. Mutant blm5 strains will be used in the proposed work to investigate the genetic and biochemical controls of oxidative damage. The blm mutants of Saccharomyces cerevisiae were isolated on the basis of their hypersensitivities to lethal effects of oxidants.
Specific aims i nclude studies of the processing and repair of DNA lesions produced after exposure of cells to oxidative DNA-damaging agents, and establishing the role of the BLM5 gene in maintaining normal eukaryotic DNA function and repair after oxidative damage. The studies will add new knowledge of general importance to fields of DNA repair and metabolism, and genetics. Understanding the introduction and repair of lesions after oxidative damage, moreover, will not be completed until all the major genes involved have been identified and characterized. Data will be acquired from interrelated studies at the cellular and molecular levels, using genetic characterization, molecular biology, and biochemistry. These include DNA sequence analyses, methodologies for studying DNA damage and repair, and transmission and scanning electron microscopy. DNA repair will be studied using pulsed-field electrophoresis, recombinational repair of double DNA breaks using yeast plasmids, and mitototic recombination assays. Experiments also include in vitro mutagenesis, the production and characterization of null mutation, identifying interactions with proteins using the two-hybrid system of identifying interacting proteins using the two-hybrid system of identifying interacting proteins, and cloning human homologs from genomic libraries. The genetic sophistication, molecular flexibility, and availability of mutant strains of S. cerevisiae permit these studies and technical approaches. The unique knowledge gained in this project has signification implications for cellular studies directed toward determining mechanisms of oxidant injury and repair as well as clinical cancer management.
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