Homologous Recombination & Human Cell Radiosensitivity

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Chromosome stability and resistance to ionizing radiation (IR) require the integrity of homologous recombin- ational repair (HRR), which acts on frank double-strand breaks (DSBs) produced in already replicated chromosomal regions. HRR is also crucial for restoring one-sided DSBs arising when DMAreplication 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. 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. 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 aberra- tions, and other endpoints. 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.