MAMMILIAN DOUBLE STRAND BREAK AND RECOMBINATIONAL REPAIR

这项拟议的研究的目标是测试关于 双链断裂(DSB)诱导同源和非同源DNA断裂的机制 哺乳动物细胞中的同源重组(HR和NHR)，包括野生- 类型和错配修复缺陷细胞以及在过度表达的细胞中 重组修复蛋白。重组是一种基本的生物学行为 过程中涉及到许多重要现象，如基因调控、DSB 修复、染色体易位和抗体基因组装。的确有 强有力的证据表明，基因重组和DNA修复缺陷与 钱泽尔。DNA损伤刺激重组，具有潜在的致突变作用 或致癌物质。出于技术原因，有关以下内容的大多数信息 哺乳动物细胞中的重组，特别是由 DNA损伤，源于对染色体外DNA的实验。 目前的证据表明，HR和NHR在以下方面存在显著差异 染色体外DNA与染色体DNA的比较。这项建议只涉及 更多与生物相关的染色体情况。 酵母中高度特异的I-SCEI核酸酶将用于裂解I-SCEI核酸酶。 体内重组底物中的SCEI位点。重组 底物将以特定的小鼠或人类染色体为靶点 以消除染色体环境的影响。这是一个受控系统 为了模拟辐射等绅士对DNA损伤诱导的重组， 化学物质，或内源核酸酶。机械论研究依赖于 底物和产物分子的结构比较通过 使用I-SCEI核酸酶，因为重组起始点 精确的实验控制，这减少了可能的 从底物到产物的路径，从而大大简化了 对结果的解释。将使用相关的重组底物 以便利在个别项目内部和之间进行比较分析。 将进行物理和遗传分析，以确定 DSB诱导的致病机制、遗传后果和遗传控制 重组修复。 具体目标1涉及关于DSB引起的人力资源机制的问题， 基因转化途径结构的研究；基因转化的关联性 和交换；同源长度和连锁要求；以及 失配修复。具体目标2涉及人力资源和国家人力资源的相对比率 在染色体DSB修复过程中，以及机械方面，如 同源中断对相对心率和近心率的影响。非- 选择性化验将提供对HR和 由DSB维修产生的NHR产品。具体目标3涉及 错配修复蛋白和重组修复蛋白作用的研究 在DSB诱导的HR期间。这些项目将极大地改善我们的 了解哺乳动物染色体中DSB的体内修复，并有 与维持基因组稳定性的相关性，特别是在细胞中 暴露在产生DSB的DNA损伤剂中，如电离 辐射。

The goal of the proposed research is to test hypotheses about the mechanism of double-strand break (DSB)-induced homologous and non- homologous recombination (HR and NHR) in mammalian cells, including wild- type and mismatch repair-deficient cells, and in cells over-expressing recombinational repair proteins. Recombination is a fundamental biological process involved in many important phenomena, such as gene regulation, DSB repair, chromosomal translocations, and antibody gene assembly. There is strong evidence linking genetic recombination and defects in DNA repair to chancer. DNA damage stimulates recombination that is potentially mutagenic or carcinogenic. For technical reasons, most information about recombination in mammalian cells, particularly recombination induced by DNA damage, was derived from experiments with extrachromosomal DNA. Current evidence indicates that both HR and NHR differ markedly in extrachromosomal versus chromosomal DNA. This proposal concerns only the more biologically relevant chromosomal situation. The highly specific I-SceI nuclease from yeast will be used to cleave I- SceI sites within recombination substrates in vivo. Recombination substrates will be targeted to a specific mouse or human chromosomal locus to eliminate chromosome environment effects. This is a controlled system for modeling DNA damage-induced recombination by gents such as radiation, chemicals, or endogenous nucleases. Mechanistic studies relying on structural comparisons of substrate and product molecules are enhanced by using I-SceI nuclease since recombination initiation sites are under precise experimental control, which reduces the number of possible pathways from substrate to product and thus greatly simplifies the interpretation of results. Relating recombination substrates will be used to facilitate comparative analysis within and among individual projects. Both physical and genetic analyses will be performed to define the mechanisms, genetic consequences, and genetic control of DSB-induced recombinational repair. Specific Aim 1 addresses questions about DSB-induced HR mechanisms, with studies of gene conversion tract structure; association of gene conversion and crossing over; homology length and linkage requirements; and roles of mismatch repair. Specific Aim 2 concerns the relative rates of HR and NHR during chromosomal DSB repair, as well as mechanistic aspects such as the effects of homology interruptions on relative HR and NHR rates. Non- selective assays will provide a comprehensive view of the range of HR and NHR products that result from DSB repair. Specific Aim 3 involves the studies of the roles of mismatch and recombinational repair proteins during DSB-induced HR. These projects will greatly improve our understanding of in vivo DSB repair in mammalian chromosomes, and have relevance to the maintenance of genomic stability, particularly in cells exposed to DNA damaging agents that produce DSBs, such as ionizing radiation.