REPAIR OF DNA DAMAGED BY UV IRRADIATION IN YEAST

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Our specific aims are to characterize the molecular mechanisms and genetic control of cellular repair of DNA damaged by ultraviolet light (UV) irradiation in the eukaryote, the yeast Saccharomyces cerevisieae. Among mutants involved in removal of UV induced p]yrimidine dimers, incision mutants will be distinguished from excision mutants by monitoring the accumulation of single strand breaks in DNA following UV irradiation in excision and ligase defective rad cdc9 double mutants. Breaks will be measured by sedimentation of DNA obtained from spheroplasts lysed by layering directly on alkaline sucrose gradients. The extent of UV-stimulated repair replication will be measured by density labeling with deoxybromouridine monophosphate (dBrUMP) in tup tmp strains. We will purify and study 5' greater than 3' exonuclease and DNA ligase, two enzymatic activities involved in excision repair, from both wild type and various repair mutants. Studies of post-replication repair will include determining the role of recombination, which genes are required for post replications repair, and whether it occurs in mitochondiral DNA. The RAD6 gene, which plays an important role in post-replication repair of UV damage and in UV mutagenesis, will be cloned by complementation of rad6-1 for UV resistance. We will then identify, purify and characterize the RAD6 protein in order to gain some insight as to how it functions in DNA repair. For determining if the same genes that affect UV mutagenesis of nuclear DNA also affect UV mutagenesis of mitochondrial DNA, we will measure the UV induced frequency of mitochondiral erythromycin resistant mutants in wild type and various repair mutants defective in UV mutagenesis of nuclear genes. Several human genetic diseases are associated with defective DNA repair and enhanced neoplastic transformation. A thorough understanding of the molecular mechanisms of DNA repair may provide a better understanding of the causes of carcinogenesis.