The long term objective is to understand the molecular mechanisms and genetic control of post-replication repair of DNA damaged by ultraviolet light (UV) in the eukaryote, Saccharomyces cerevisiae. The following studies will be carried out to achieve this goal. The RAD6 gene, which is required for postreplication repair, induced mutagenesis, and other important cellular functions will be randomly mutagenized to generate various mutations and also to isolate temperature sensitive (ts) mutants of each of the three major phenotypes associated with rad6 mutants: UV sensitivity, UV immutability, and sporulation deficiency. Site-specific mutagenesis will be used to delete and modify the polyacidic tract in the carbosyl-terminal region of the RAD6 protein to determine its function. The role of the RAD6 and other genes in postreplication repair will be assessed from in vivo experiments utilizing UV irradiated single-stranded plasmids as well as from in vitro experiments to measure bypass replication. The purified RAD6 protein will be further characterized by determining whether it interacts with nucleosomes. Since the RAD6 gene is induced following UV irradiation and also during the cell cycle, the DNA sequences in the RAD6 gene required for its induction will be determined. A single protein in Schizosaccharomyces pombe cross-reacts with antibody directed against the RAD6 protein of S. cerevisiae. The S. pombe gene encoding this protein will be cloned and the protein encoded by it will be characterized. Further extension of studies on the molecular mechanisms of postreplication repair will include isolation and characterization of the RAD18, REV3, and RAD9 genes, the other important genes in the RAD6 epistasis group. The proteins encoded by these genes will be purified and studied. For attaining the ultimate goal of defining the components of the protein complexes involved in postreplication repair and induced mutagenesis, proteins or genes encoding proteins which interact with RAD6 protein will be isolated directly using either a genetic approach or a biochemical approach. The genetic approach will entail isolation of cold sensitive suppressors of heat sensitive UV sensitive or UV immutable rad6 mutants. The biochemical approach will involve immunoprecipitation of proteins associated with RAD6 in a complex and affinity chromatography of proteins which bind to RAD6. In addition, suppresors of the UV sensitivity or UV immutability of rad6 deletions will be isolated and the genetic mechanism(s) of suppression identified and characterized. Such bypass suppression can uncover new genes which can overcome the rad6 deletion defect. Defective DNA repair and enhanced neoplasia characterize several human genetic diseases. A thorough understanding of the molecular mechanisms of DNA repair may provide a better understanding of the causes of carcinogenesis.
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