The goal of the proposed research is to determine the structural and enzymatic roles of several mammalian proteins in homologous recombinational repair (HR) of DNA double-strand breaks (DSBs), and to determine how these proteins regulate spontaneous HR. DSBs are induced by chemicals and radiation and they arise spontaneously during DNA replication. DSBs are repaired by HR and by nonhomologous end-joining (NHEJ). Proteins with direct roles in HR include RAD51, five RAD51 paralogs (XRCC2, XRCC3, RAD51B, RAD51C, and RAD51D), BRCA1, and BRCA2. RAD51 catalyzes DNA strand transfer and is therefore central to HR processes. However, RAD51 is difficult to study in mammalian cells because null mutants are cell- and embryo-lethal. The RAD51 paralogs form several complexes, and these complexes physically interact with RAD51 and/or DNA, and are thought to augment RAD51 activity. BRCA2 interacts with RAD51 and other proteins, as well as single- and double-stranded DNA. The RAD51 paralogs and BRCA2 are thus uniquely positioned to regulate HR efficiency and outcome. Defects and/ or altered expression of any of these proteins result in genome instability and cancer predisposition. We recently demonstrated that XRCC3 influences early and late stages of HR, and that several BRCA2 functional domains have dominant negative effects on DSB-induced HR in human cells.
Two specific aims are proposed to investigate (1) the structural and enzymatic roles of XRCC2, XRCC3, and RAD51C in DSB-induced and spontaneous HR; and (2) the roles of several functional domains of BRCA2 in DSB-induced and spontaneous HR, including BRCA2 domains that interact with single- and double-stranded DNA, RAD51, and other proteins. These projects will help establish roles for specific structural or enzymatic domains of XRCC2, XRCC3, RAD51C, and BRCA2 in early and/or late stages of HR, and thereby provide new insight into the regulation of HR, maintenance of genome stability, and tumor suppression. Because DSB repair is a key determinant of cell survival following DNA damage, the information gained from these studies will also facilitate the development of more effective agents to sensitize tumor cells to DNA damage by radiation and genotoxic chemicals. Studies of HR mechanisms and HR regulatory proteins will also facilitate the development of more effective gene targeting and gene therapy systems.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
3R01CA077693-07S1
Application #
7123263
Study Section
Special Emphasis Panel (ZRG1)
Program Officer
Ogunbiyi, Peter
Project Start
2005-08-01
Project End
2007-07-31
Budget Start
2005-08-01
Budget End
2005-12-31
Support Year
7
Fiscal Year
2005
Total Cost
$20,378
Indirect Cost
Name
University of New Mexico
Department
Microbiology/Immun/Virology
Type
Schools of Medicine
DUNS #
868853094
City
Albuquerque
State
NM
Country
United States
Zip Code
87131
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Lu, Huimei; Yue, Jingyin; Meng, Xiangbing et al. (2007) BCCIP regulates homologous recombination by distinct domains and suppresses spontaneous DNA damage. Nucleic Acids Res 35:7160-70
Huang, Lei; Kim, Perry M; Nickoloff, Jac A et al. (2007) Targeted and nontargeted effects of low-dose ionizing radiation on delayed genomic instability in human cells. Cancer Res 67:1099-104
Durant, Stephen T; Paffett, Kimberly S; Shrivastav, Meena et al. (2006) UV radiation induces delayed hyperrecombination associated with hypermutation in human cells. Mol Cell Biol 26:6047-55
Schildkraut, Ezra; Miller, Cheryl A; Nickoloff, Jac A (2006) Transcription of a donor enhances its use during double-strand break-induced gene conversion in human cells. Mol Cell Biol 26:3098-105
Lu, Huimei; Guo, Xu; Meng, Xiangbing et al. (2005) The BRCA2-interacting protein BCCIP functions in RAD51 and BRCA2 focus formation and homologous recombinational repair. Mol Cell Biol 25:1949-57
Schildkraut, Ezra; Miller, Cheryl A; Nickoloff, Jac A (2005) Gene conversion and deletion frequencies during double-strand break repair in human cells are controlled by the distance between direct repeats. Nucleic Acids Res 33:1574-80
Bailey, Susan M; Brenneman, Mark A; Halbrook, James et al. (2004) The kinase activity of DNA-PK is required to protect mammalian telomeres. DNA Repair (Amst) 3:225-33
Huang, Lei; Grim, Suzanne; Smith, Leslie E et al. (2004) Ionizing radiation induces delayed hyperrecombination in Mammalian cells. Mol Cell Biol 24:5060-8
Bailey, Susan M; Brenneman, Mark A; Goodwin, Edwin H (2004) Frequent recombination in telomeric DNA may extend the proliferative life of telomerase-negative cells. Nucleic Acids Res 32:3743-51

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