Mammalian Replication fork stalling

Beth Israel Deaconess Medical Center, Boston MA

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Abstract

DESCRIPTION (provided by applicant): The aim of this proposal is to develop novel tools for studying the relationship between stalled or broken replication forks and sister chromatid recombination in mammalian cells. Although current models propose that recombination at sites of stalled DNA synthesis is critical for preventing genomic instability and cancer, the ability to model this process in mammalian cells is severely limited, due to the absence of recombination reporters that can accurately model this process in a quantitative and mechanistic fashion. Work proposed herein will provide such tools, by combining "state-of-the-art" mammalian reporters of homologous recombination and sister chromatid recombination with novel strategies to cause site-specific replication fork stalling. This work will provide a much needed new tool to study this area of critical importance for human cancer predisposition. Our specific aims are: 1. Adapt a known replication fork barrier for the induction of site-specific HR in mammalian cells. 2. Study HR and SCR at a replication fork barrier in mammalian cells. 3. Develop "nickase" mutants of I-SceI to stimulate HR and SCR at a chromosomal I-SceI site in mammalian cells.