Homologous Recombination & Human Cell Radiosensitivity

同源重组

• 批准号: 7386707
• 负责人: LAWRENCE H THOMPSON
• 金额: $ 34.35万
• 依托单位: UNIVERSITY OF CALIF-LAWRNC LVRMR NAT LAB
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-02-07 至 2009-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7386707
• 关键词: B-Lymphocytes; BRCA2 gene; Biochemistry; Biological; Bypass; Cell Cycle; Cell Line; Cell Survival; Cells; Chicken Cells; Chickens; Chinese Hamster Ovary Cell; Chromosomal Instability; Chromosomal Stability; Chromosome abnormality; Class; DHFR gene; Dependence; Diploidy; Disease regression; Double Strand Break Repair; End Point; Fanconi' s Anemia; Fibroblasts; Figs - dietary; G1 Phase; Gene Amplification; Gene Mutation; Gene Rearrangement; Gene Targeting; Genetic; Genome Stability; Genomic Instability; Genomics; Genotype; Hamsters; Human; Human Cell Line; Ionizing radiation; Knock-out; Knowledge; Lead; Lesion; Malignant Neoplasms; Mammalian Cell; Measures; Molecular; Mutagenesis; Mutation; Mutation Spectra; Nature; Pathway interactions; Phase; Phenotype; Play; Point Mutation; Process; Proteins; Radiation Tolerance; Radiation therapy; Radiation, Other; Rate; Research Personnel; Resistance; Rodent; Role; Side; Sister Chromatid; Sister Chromatid Exchange; System; Testing; Variant; XRCC3 gene; base; cell killing; cell type; concept; defined contribution; homologous recombination; insight; irradiation; killings; mutant; null mutation; paralogous gene; prevent; programs; radiation resistance; recombinational repair; repaired

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.

染色体的稳定性和对电离辐射（IR）的抗性需要同源重组的完整性。 正常修复（HRR），它作用于已经复制的细胞中产生的坦率的双链断裂（DSB）。 染色体区域HRR对于恢复DMA复制分叉时产生的单侧DSB也至关重要 崩溃，甚至可以通过促进非诱变性的阻断旁路来拯救被阻断的分叉 氧化损伤在非人类脊椎动物细胞中，Rad 51旁系同源物（XRCC 2/3 & Rad 51 B/C/D）中的突变 赋予相似的表型，中等IR敏感性和高染色体不稳定性。因此，这五个 蛋白质为研究HRR的分子性质提供了框架。这个项目使用基因敲除突变体 CHO仓鼠和人类细胞系，以确定HRR促进染色体稳定性的机制 和抗辐射性。AIM 1检验了HRR抑制癌症相关类型的 遗传改变，如通过表征rad 51 d敲除CHO细胞所揭示的。基因突变率 将定量dhfr和CAD基因座的hprt基因座和基因扩增，并将hprt突变谱 将被定性。AIM 2将在Tp 53正常永生化二倍体中构建XRCC 3的无效突变株 人成纤维细胞和评估其基因组不稳定性方面的放射敏感性，染色体畸变， 以及其他端点。AIM 3将确定HRR对IR电阻变化的贡献， 仓鼠和人类细胞的细胞周期。在这个目标中要检验的假设是：（a）经典S相 抗性是由于HRR;（B）HRR有助于G1期照射的细胞在未修复时的存活 损伤随后由DMA复制机制处理;（c）IR诱导的hprt突变的产量是 在S期，HRR比G1期低，这是因为HRR在S期起作用，促进DSB的无错误修复。 这些综合研究可能会为癌症放射治疗提供更合理的基础，并为癌症的治疗带来更多的见解。 HRR如何通过内源性过程和外源性因子阻止癌发生的启动。