Chromosome stability and resistance to ionizing radiation (IR) require the integrity of homologous recombine- national repair (HRR), which acts on frank double-strand breaks (DSBs) produced in the already replicated chromosomal regions. HRR is also crucial for restoring one-sided DSBs arising when DMA replication forks collapse, and may even act by rescuing blocked forks by facilitating non-mutagenic bypass of blocking oxidative lesions. In non-human vertebrate cells, mutations in the Rad51 paralogs (XRCC2/3 & Rad51B/C/D) confer similar phenotypes of moderate IR sensitivity and high chromosome instability. Thus, these five proteins provide a framework for studying the molecular nature of HRR. This project uses knockout mutants of CHO hamster and human cell lines to identify mechanisms by which HRR promotes chromosome stability and radiation resistance.
Specific Aim 1 tests the hypothesis that HRR suppresses cancer-associated types of genetic alterations, as revealed by characterizing Rad51d knockout CHO cells. Rates of gene mutation at the hprt locus and gene amplification at the dhfr and CAD loci will be quantified, and the hprt mutation spectrum will be characterized.
Specific Aim 2 will construct null mutant lines of XRCC3 in Tp53-normal immortalized diploid human fibroblasts and assess their genomic instability with respect to radiosensitivity, chromosomal aberrations, and other endpoints.
Specific Aim 3 will determine the contribution of HRR to changes in IR resistance during the cell cycle in hamster and human cells. The hypotheses to be tested in this aim are: (a) Classical S phase resistance is due to HRR; (b) HRR contributes to the survival of cells irradiated in G1 phase when unrepaired damage is later processed by the DMA replication machinery; (c) The yield of IR-induced hprt mutations is lower in S phase than in G1 phase because HRR acts during S phase to promote error-free repair of DSBs. These integrated studies may lead to a more rational basis for cancer radiotherapy and bring insights into how HRR prevents the initiation of carcihogenesis by endogenous processes and exogenous agents. ? ?

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
1R01CA112566-01A1
Application #
7047272
Study Section
Radiation Therapeutics and Biology Study Section (RTB)
Program Officer
Pelroy, Richard
Project Start
2006-02-07
Project End
2010-01-31
Budget Start
2006-02-07
Budget End
2007-01-31
Support Year
1
Fiscal Year
2006
Total Cost
$353,750
Indirect Cost
Name
Lawrence Livermore National Laboratory
Department
Biology
Type
Organized Research Units
DUNS #
827171463
City
Livermore
State
CA
Country
United States
Zip Code
94550
Thompson, Larry H (2012) Recognition, signaling, and repair of DNA double-strand breaks produced by ionizing radiation in mammalian cells: the molecular choreography. Mutat Res 751:158-246
Thompson, Larry H (2012) Losing and finding myself in DNA repair. DNA Repair (Amst) 11:637-48
Urbin, Salustra S; Elvers, Ingegerd; Hinz, John M et al. (2012) Uncoupling of RAD51 focus formation and cell survival after replication fork stalling in RAD51D null CHO cells. Environ Mol Mutagen 53:114-24
Thompson, Larry H; Jones, Nigel J (2010) Stabilizing and remodeling the blocked DNA replication fork: anchoring FANCM and the Fanconi anemia damage response. Mol Cell 37:749-51
Wilson, Paul F; Hinz, John M; Urbin, Salustra S et al. (2010) Influence of homologous recombinational repair on cell survival and chromosomal aberration induction during the cell cycle in gamma-irradiated CHO cells. DNA Repair (Amst) 9:737-44
Rybanska, Ivana; Gursky, Jan; Faskova, Miriam et al. (2010) Newly identified CHO ERCC3/XPB mutations and phenotype characterization. Mutagenesis 25:179-85
Hinz, John M; Urbin, Salustra S; Thompson, Larry H (2009) RAD51D- and FANCG-dependent base substitution mutagenesis at the ATP1A1 locus in mammalian cells. Mutat Res 665:61-6
Thompson, Larry H; Hinz, John M (2009) Cellular and molecular consequences of defective Fanconi anemia proteins in replication-coupled DNA repair: mechanistic insights. Mutat Res 668:54-72
Nagasawa, Hatsumi; Wilson, Paul F; Chen, David J et al. (2008) Low doses of alpha particles do not induce sister chromatid exchanges in bystander Chinese hamster cells defective in homologous recombination. DNA Repair (Amst) 7:515-22
Hinz, John M; Nham, Peter B; Urbin, Salustra S et al. (2007) Disparate contributions of the Fanconi anemia pathway and homologous recombination in preventing spontaneous mutagenesis. Nucleic Acids Res 35:3733-40

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