The goal of our research is to provide a cellular and molecular understanding of human Rad51-mediated DNA repair. The catalytic activity of Rad51 is of fundamental importance to homologous recombination, a process required for maintenance of genome integrity under both normal metabolic conditions and in response to mutagenic stress. Studies in this revised proposal are directed at understanding how Rad51 function is regulated through specific protein-protein interactions. In revised Aim 1 we will use RNAi and cell-based methods to explore the functional relationship of Rad51 with other proteins known to assist its cellular activity. We have found that Rad51 stability and degradation are regulated through its interaction with the Rad51 paralog protein, Rad51C. Additionally, our data suggest that Rad51 degradation is compartmentalized within the cell, and work in this Aim will address how this is regulated. Work in revised Aim 3 focuses on analysis of specific mutant proteins to advance our understanding of the biochemical processes that underlie Rad51-mediated DNA repair. We will take advantage of our recently published methodology using RNAi to analyze the function of mutant Rad51, Rad51C and Xrcc3 proteins in human cells. Biochemical studies of mutant proteins will also be included to provide further mechanistic insight into the cellular requirements of specific protein functions.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
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Molecular Genetics B Study Section (MGB)
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Hagan, Ann A
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University of Massachusetts Medical School Worcester
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Sage, Jay M; Knight, Kendall L (2013) Human Rad51 promotes mitochondrial DNA synthesis under conditions of increased replication stress. Mitochondrion 13:350-6
Sage, Jay M; Gildemeister, Otto S; Knight, Kendall L (2010) Discovery of a novel function for human Rad51: maintenance of the mitochondrial genome. J Biol Chem 285:18984-90
Bakhlanova, Irina V; Dudkina, Alexandra V; Baitin, Dima M et al. (2010) Modulating cellular recombination potential through alterations in RecA structure and regulation. Mol Microbiol 78:1523-38
Gildemeister, Otto S; Sage, Jay M; Knight, Kendall L (2009) Cellular redistribution of Rad51 in response to DNA damage: novel role for Rad51C. J Biol Chem 284:31945-52
Bennett, Brian T; Bewersdorf, Jorg; Knight, Kendall L (2009) Immunofluorescence imaging of DNA damage response proteins: optimizing protocols for super-resolution microscopy. Methods 48:63-71
Forget, Anthony L; Kudron, Michelle M; McGrew, Dharia A et al. (2006) RecA dimers serve as a functional unit for assembly of active nucleoprotein filaments. Biochemistry 45:13537-42
Forget, Anthony L; Bennett, Brian T; Knight, Kendall L (2004) Xrcc3 is recruited to DNA double strand breaks early and independent of Rad51. J Cell Biochem 93:429-36
Nastri, H G; Knight, K L (1994) Identification of residues in the L1 region of the RecA protein which are important to recombination or coprotease activities. J Biol Chem 269:26311-22
Konola, J T; Logan, K M; Knight, K L (1994) Functional characterization of residues in the P-loop motif of the RecA protein ATP binding site. J Mol Biol 237:20-34
Skiba, M C; Knight, K L (1994) Functionally important residues at a subunit interface site in the RecA protein from Escherichia coli. J Biol Chem 269:3823-8

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