REPAIR OF DNA DAMAGED BY UV IRRADIATION IN YEAST

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The long term objectives of this proposal are to understand the molecular mechanisms of bypass replication of UV damaged DNA templates in the yeast Saccharomyces cerevisiae. Xeroderma pigmentosum patients suffer from a high incidence of skin cancers, presumably because of the mutagenic bypass of unexcised UV lesions from DNA. The proposed project will focus on the predominantly error-free branch of the postreplication repair pathway in yeast. This pathway is of considerable importance because it generates a relatively low level of mutations compared to the mutagenic bypass pathway. Genetic and biochemical studies will be done to identify the various components of the error free bypass pathway, to define their biochemical activities, and to reconstitute bypass replication in vitro. Postreplication repair will be examined in mutants of the RAD5 and PCNA genes, singly and in combination with rev3delta. The involvement of DNA polymerases delta and epsilon and of single strand binding protein RFA in postreplication repair will be ascertained by genetic and molecular studies. The domains that mediate interaction of Rad6 and Rad 18 proteins will be defined and the structure of interacting peptides determined by NMR spectroscopy. The role of Rad 18 ATPase in DNA binding and in turnover of the Rad6/Rad 18 complex will be examined, and DNA binding activity of the complex will be further characterized. The biological significance of ATP binding and hydrolysis will be ascertained by mutating the lysine residue in the GKS motif of Rad 18 and examining its effect on DNA repair. Mutant rad18 proteins will be purified and examined for their ability to bind and hydrolyze ATP, and for DNA binding. The role of the Rad 18 zinc finger domain in DNA binding will be examined by site directed mutagenesis of specific residues of this motif and determination of the effect of these rad18 mutations on DNA repair and on DNA binding. Binding of Rad5 to different DNA substrates will be examined, and studies will be done to determine if Rad6/Rad 18 and Rad5 are loaded onto the DNA replication machinery on damaged DNA. Protein-protein interactions among Rad6, Radl8, Rad5, PCNA, and other proteins found to have a role in postreplicative gap filling will be studied by coimmunoprecipitation, by affinity chromatography, and by the two hybrid system. Genes encoding other proteins that interact with Rad6, Rad18, and Rad5 will be cloned by the two hybrid method. This method should uncover any additional proteins that may be required for bypass replication, as well as the in vivo substrates of Rad6. Whether Rad6/Radl8 complex mediates the ubiquitination and subsequent degradation or proteolytic cleavage of specific repair or replication proteins following treatment of cells with DNA damaging agents will be determined. Cell-free extracts from RAD+ and various repair deficient mutants will be examined for bypass replication of UV irradiated DNA substrates. Bypass replication of templates carrying a site specific UV lesion will be reconstituted using purified components.