DNA recombination is an essential process that repairs DNA double-strand breaks (DSBs) and gaps that occur spontaneously, or are induced by chemicals or irradiation. In human, defects in recombination result in immune deficiencies, infertility, neurodegenerative disorders, developmental abnormalities, aging and cancer. DSBs as the most cytotoxic lesions are a fundamental component of the most prevalent cancer treatments, radiotherapy and radiomimetic 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 a template DNA molecule. The goal of this project is to understand the mechanisms and regulation of DNA synthesis during recombination, which remains very poorly understood. Two assays will be utilized to examine repair DNA synthesis that reflect two major recombination pathways with distinct DNA synthesis features. Both pathways play different yet important roles in cells. One is the simple repair of two-ended DSBs by gene conversion where both 3' ends prime DNA synthesis and thus only short leading strands are synthesized. The second assay employs 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 a mechanism 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 redundant functions of multiple DNA polymerases recruited to DSBs and determine which DNA helicases and other enzymes specifically promote DNA synthesis during homologous recombination. The major focus will be on studying Pif1, the DNA helicase that we propose to be the first eukaryotic nonreplicative helicase that stimulates DNA synthesis during recombination. (2) To understand the mechanism of Break Induced Replication. Using isotope density transfer we will establish the mode of DNA synthesis in BIR and determine the role of DNA helicases and structure specific nucleases in BIR. Together we will provide a comprehensive view of DNA synthesis during homologous recombination.
This proposal aims to understand the molecular mechanisms and regulation of DNA recombination, an essential DNA repair pathway preventing genome instability and cancer. Both the mechanism and proteins mediating DNA repair processes are conserved in evolution, so our proposed research in the model organism.
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