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Regulation of DNA recombination in Saccharomyces cerevisiae

Grzegorz A. Ira

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National Institutes of Health (NIH)
DNA recombination is an essential process that repairs DNA double-strand breaks (DSBs) andgaps that occur spontaneously, or are induced by chemicals or irradiation. In human, defects inrecombination result in immune deficiencies, infertility, neurodegenerative disorders,developmental abnormalities, aging and cancer. DSBs as the most cytotoxic lesions are afundamental component of the most prevalent cancer treatments, radiotherapy andradiomimetic chemotherapy. Therefore defining the genetic requirements and mechanisms of recombination pathways is of critical importance. A fundamental reaction during repair of broken chromosomes by recombination is DNA synthesis that copies homologous sequences from atemplate DNA molecule. The goal of this project is to understand the mechanisms andregulation of DNA synthesis during recombination, which remains very poorly understood. Two assays will be utilized to examine repair DNA synthesis that reflect two major recombinationpathways with distinct DNA synthesis features. Both pathways play different yet important rolesin cells. One is the simple repair of two-ended DSBs by gene conversion where both 3' endsprime DNA synthesis and thus only short leading strands are synthesized. The second assayemploys break induced replication (BIR), in which a single DSB end invades a template,followed by extensive leading- and lagging- strand DNA synthesis. BIR is thought to be amechanism of HR-dependent telomere maintenance in the absence of telomerase found in 10-15% of all cancers. The specific aims are: (1) To understand the unique and redundantfunctions of multiple DNA polymerases recruited to DSBs and determine which DNA helicasesand other enzymes specifically promote DNA synthesis during homologous recombination. Themajor focus will be on studying Pif1, the DNA helicase that we propose to be the first eukaryoticnonreplicative helicase that stimulates DNA synthesis during recombination. (2) To understandthe mechanism of Break Induced Replication. Using isotope density transfer we will establishthe mode of DNA synthesis in BIR and determine the role of DNA helicases and structurespecific nucleases in BIR. Together we will provide a comprehensive view of DNA synthesisduring homologous recombination.

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