The biological effects of UV light irradiation of cells include death, mutation and neoplastic transformation. DNA is the biologically relevant target of UV light exposure, resulting in the formation of primarily cyclobutane pyrimidine dimers (CPDs) and (6-4) photoproducts (6-4 PPs). CPDs and 6-4 PPs have been shown to be cytotoxic, mutagenic, and carcinogenic in cell and animal model systems and are readily formed in human skin cells following exposure to sunlight. Skin cancer, the most frequently occurring cancer in humans, is primarily associated with chronic exposure to solar radiation. The majority of both basal and squamous cell carcinomas of the skin contain mutations in the p53 tumor suppressor gene and are of the type caused by CPDs and 6-4 PPs. An understanding of the components and biochemistry of the repair of CPDs and 6-4 PPS in eukaryotes should provide important insights into the mechanisms of UV carcinogenesis. The fission yeast, Schizosaccharomyces pombe provides an attractive biochemical and genetic eukaryotic DNA repair model system with features that include an abundant source of cells for enzyme purification and characterization, relatively straightforward genetic dissectability, and the characterizing a S. pombe endonuclease that specifically recognizes both CPDs and 6-4 PPs and makes direct 5'incisions at the sites of these UV photoproducts. We have biological and genetic evidence that this enzyme, S. pombe DNA endonuclease (SPDE), represents the initial step of an alternative DNA excision repair system which we have termed the SPDE-dependent DNA repair (SDR) pathway. It now appears that SDR-like pathways are also present in other organisms. The goal of the proposed research will be to characterize the SDR pathway with a significant focus on SPDE. A combination of biochemical and molecular biological approaches will be taken to provide direct information on SPDE and the SDR pathway. The major objectives will be to (1) complete the purification and conduct a detailed enzymological characterization of SPDE; (2) identify and characterize the gene encoding SPDE; (3) identify and characterize other components of the SDR pathway; and (4) screen a selection of candidate organisms, including humans, for a SPDE-like activity to determine the species distribution of the SDR pathway. The information obtained in these studies should greatly increase our understanding of the eukaryotic cellular machinery that reverses carcinogenic UV photoproducts.
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