Homologous Recombination & Human Cell Radiosensitivity

描述（由申请方提供）：染色体稳定性和对电离辐射（IR）的耐受性需要同源重组-国家修复（HRR）的完整性，其作用于已复制染色体区域中产生的明显双链断裂（DSB）。HRR对于恢复DMA复制叉崩溃时产生的单侧DSB也是至关重要的，甚至可以通过促进阻断氧化损伤的非诱变旁路来拯救阻断的叉。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）中的突变赋予了中等IR敏感性和高染色体不稳定性的相似表型。因此，这五种蛋白质为研究HRR的分子性质提供了框架。本项目使用CHO仓鼠和人类细胞系的敲除突变体来鉴定HRR促进染色体稳定性和辐射抗性的机制。特定目的1测试HRR抑制癌症相关类型的遗传改变的假设，如通过表征Rad 51 d敲除CHO细胞所揭示的。将定量hprt基因座的基因突变率以及dhfr和CAD基因座的基因扩增率，并表征hprt突变谱。Specific Aim 2将在Tp 53正常永生化二倍体人成纤维细胞中构建XRCC 3的无效突变系，并评估其基因组在放射敏感性、染色体畸变和其他终点方面的不稳定性。具体目标3将确定HRR对仓鼠和人细胞细胞周期期间IR抗性变化的贡献。在这个目标中要测试的假设是：（a）经典的S期抗性是由于HRR;（B）HRR有助于在G1期照射的细胞的存活，当未修复的损伤随后被DMA复制机器处理时;（c）IR诱导的hprt突变的产量在S期比在G1期低，因为HRR在S期期间起作用以促进DSB的无错误修复。这些综合研究可能会导致一个更合理的基础上，癌症放疗，并带来的见解HRR如何防止启动致癌的内源性过程和外源性药物。

DESCRIPTION (provided by applicant): 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.