Cancer is a leading cause of death and morbidity. It results from the accumulation of genetic mutations that ultimately lead to a growth advantage and expansion of rare cellular clones. Understanding the etiology of genetic mutation is thus of tremendous importance. Fundamentally, mutations represent the failure of a cell to correctly repair the DNA lesions caused by a variety of endogenous and exogenous damaging agents. This proposal is based on the model that inefficient repair of DNA double-strand breaks (DSBs) leads to persistence of lesions that ultimately become substrates for chromosomal rearrangement, a hallmark of malignancy. It focuses specifically on the enzymes that process DSB ends to make them ready for ligation, since failure of these enzymes might contribute substantially to persistence of DSB lesions in recombinogenic forms. All experiments use Saccharomyces cerevisiae as a model organism. State-of-the-art genomic tools are used to study many yeast genes in parallel and in combination, which is critical since multiplicity and redundancy of end processing pathways is anticipated. The first Specific Aim is to identify enzymes that resect the 5'-terminated strand in homologous recombinational repair (HRR). Preliminary evidence suggests that these cause an efficient and irreversible commitment to HRR in budding yeast. Novel competitive assays are based on the hypothesis that impaired 5' resection will increase the contribution of nonhomologous end-joining (NHEJ) and the likelihood of chromosomal rearrangement. High probability candidate helicases and nucleases will be examined in detail, in addition to panel and mutational screens for other involved genes. The second Specific Aim is to elucidate the mechanism of POL4 (yeast DNA polymerase b)-dependent processing in NHEJ. The hypothesis that this gene's previously described role represents an overlap with base excision repair is examined by chimeric analysis with the human enzyme. Protein interaction, panel and mutational screens will be used to identify Po14p-interacting nucleases. The third Specific Aim is to describe the processing of 5' hydroxyl and 3' phosphate terminal DSB lesions. A novel plasmid transformation assay will be used to explore the extent and mechanism of repair. This assay, in vitro biochemical assays, and cellular responses to chemical mutagens will evaluate the hypothesis that ORF YMR156c is the 3' phosphatase portion of yeast polynucleotide kinase.
