MAMMILIAN DOUBLE STRAND BREAK AND RECOMBINATIONAL REPAIR

拟议研究的目标是测试有关 双链断裂（DSB）诱导的同源和非同源 哺乳动物细胞中的同源重组（HR和NHR），包括野生型- 型和错配修复缺陷的细胞，并在细胞过度表达 重组修复蛋白。新陈代谢是一种基本的 这一过程涉及许多重要现象，如基因调控、DSB 修复、染色体易位和抗体基因组装。有 强有力的证据将基因重组和DNA修复缺陷联系起来， 投机者DNA损伤刺激重组，这是潜在的诱变 或致癌。由于技术原因，大多数关于 哺乳动物细胞中的重组，特别是由 DNA损伤，来自染色体外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和染色体DNA这项建议只涉及 更有生物学意义的染色体情况。 来自酵母的高度特异性的I-SceI核酸酶将用于切割I-SceI。 体内重组底物内的SceI位点。重组 底物将靶向特定的小鼠或人染色体基因座 以消除染色体环境影响。这是一个受控系统 为了模拟由辐射等辐射引起的DNA损伤诱导的重组， 化学物质或内源性核酸酶。机制研究依赖于 底物和产物分子的结构比较通过 使用I-SceI核酸酶，因为重组起始位点在 精确的实验控制，减少了可能的数量 从底物到产物的途径，从而大大简化了 结果的解释。将使用相关的重组底物 以便于在各个项目内部和项目之间进行比较分析。 将进行物理和遗传分析，以确定 DSB诱导的细胞凋亡的机制、遗传后果和遗传控制 重组修复 具体目标1解决了有关DSB诱导的HR机制的问题， 基因转换域结构研究;基因转换关联 和交换;同源长度和连锁要求;和作用 错配修复具体目标2涉及HR和NHR的相对比率 在染色体DSB修复过程中，以及机械方面，如 同源性中断对相对HR和NHR率的影响。非 选择性测定将提供HR范围的全面视图， 由DSB修复产生的NHR产品。具体目标3涉及 错配和重组修复蛋白的作用研究 这些项目将大大提高我们的人力资源管理水平， 了解哺乳动物染色体中的体内DSB修复， 与维持基因组稳定性的相关性，特别是在细胞中 暴露于产生DSB的DNA损伤剂，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.