Exposure of cells to ionizing radiation, UV light or chemicals can result in DNA damage, but usually repair mechanisms are available to mend the lesions induced. However, incomplete or faulty repair can occur and can have deleterious consequences, including cell lethality ,mutagenesis, teratogenesis or carcinogenesis. Therefore, an understanding of DNA repair mechanisms, a process fundamental to all living organisms, is important. To better understand the way cells respond to radiation or chemical exposure, this project focuses on molecular aspects of DNA repair primarily in the small eukaryote Schizosaccharomyces pombe. This yeast will serve as a model system because it affords several significant advantages over the use of mammalian cells in the laboratory, yet S. pombe shares many molecular, biological and biochemical features with mammalian systems. Recently, I isolated a S. pombe mutant exhibiting temperature-dependent sensitivity to UV light and ionizing radiation. Genetic analyses indicated that a previously unidentified, altered rad locus (i.e., called rad23-1) was responsible for the phenotype observed. In this proposal, I plan to characterize the mutant further, including the determination of chemical sensitivity, mutability and recombination ability. This mutant, as well as the S. pombe rad3-136 mutant, will be the focus of ability. This mutant, as well as the S. pombe rad3-136 mutant, will be the focus of molecular studies. No S. pombe DNA repair genes have been cloned to date. I propose to isolate the wild type rad23 and rad3 genes. The corresponding gene products encoded by these loci participate in repair of DNA damage induced by both UV light and ionizing radiation since alterations within these genes cause radiation sensitivity. Furthermore, it is known that the rad3 gene mediates UV light-induced mutagenesis. The structures of these cloned genes will be determined, and their inducibility by DNA damaging agents and/or preferential expression during a specific phase of the cell cycle will be tested to learn more about their regulation. The DNA sequence changes in the mutant rad genes, relative to wild type cognates, that make them responsible for radiation and chemical sensitivity will be identified. Limited studies will also be performed to determine whether S. pombe DNA repair gene sequences are conserved in other species. Together, these studies should provide important information relating to S. pombe DNA repair and possibly cognate systems in other organisms.
