Homologous Recombination & Human Cell Radiosensitivity

染色体的稳定性和抗电离辐射(IR)要求同源重组的完整性。 状态修复(HRR)，作用于已复制的坦率双链断裂(DSB) 染色体区域。对于恢复在DMA复制分叉时产生的单边DSB，HRR也至关重要 崩溃，甚至可以通过促进阻塞的非突变旁路来拯救阻塞的叉子 氧化损伤。在非人类脊椎动物细胞中，RAD51近亲基因(XRCC2/3和RAD51B/C/D)的突变 具有中等红外线敏感度和高度染色体不稳定性的相似表型。因此，这五个人 蛋白质为研究HRR的分子性质提供了一个框架。这个项目使用的是基因敲除突变体 CHO仓鼠和人类细胞系的研究，以确定HRR促进染色体稳定性的机制 和抗辐射能力。Aim 1验证了HRR抑制癌症相关类型的 RAD51D基因敲除CHO细胞的特征揭示了遗传变化。基因突变率在 HPRT基因座和DHFR和CAD基因座的基因扩增将被量化，HPRT突变谱将被量化 将被描述为。目的2构建TP53-正常永生化二倍体中XRCC3的零突变系 人成纤维细胞，并评估它们的基因组不稳定性与辐射敏感性，染色体Aberra- 选项和其他端点。目标3将确定HRR对年内IR抵抗变化的贡献 仓鼠和人类细胞的细胞周期。在这一目标中要检验的假设是：(A)经典的S阶段 抗性是由于HRR；(B)HRR有助于处于G1期的细胞在未经修复的情况下存活 损害稍后由DMA复制机制处理；(C)IR诱导的HPRT突变的产率为 S期低于G1期，这是因为心率变异性在S期起作用，促进双链断裂的无误修复。 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 HRR如何通过内源性过程和外源性因素阻止癌变的启动。

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.