End Processing in DNA Double Strand Break Repair

描述（由申请人提供）：癌症是死亡的主要原因， 发病率它是基因突变积累的结果， 最终导致稀有细胞克隆的生长优势和扩张。 因此，了解基因突变的病因是巨大的 重要性从根本上说，突变代表了细胞的失败， 正确修复由各种内源性和 外源性损伤因子。这一建议是基于这样一种模式： DNA双链断裂（DSB）的修复导致损伤的持续， 最终成为染色体重排的底物， 恶性肿瘤它特别关注的酶，处理DSB结束， 使它们准备好连接，因为这些酶的失效可能会导致 DSB损伤在重组形式中的持续存在。所有 实验使用酿酒酵母（Saccharomyces cerevisiae）作为模式生物。State-of-the-art 基因组工具被用来研究许多酵母基因在平行和 组合，这是至关重要的，因为末端的多重性和冗余性 处理路径是预期的。第一个具体目标是确定 在同源重组中切除5 '-末端链的酶 修复（HRR）。初步证据表明，这些导致了一个有效的， 不可逆的承诺HRR在芽殖酵母。新的竞争性测定是 基于受损的5'切除将增加 非同源末端连接（NHEJ）的贡献和 染色体重排高概率候选解旋酶和核酸酶 将详细检查，除了面板和突变筛选， 其他相关基因第二个具体目标是阐明 NHEJ中的POL 4（酵母DNA聚合酶B）依赖性加工。的假设 该基因先前描述的作用与碱基切除重叠 通过用人酶的嵌合分析来检查修复。蛋白 将使用交互作用、面板和突变筛选来鉴定 Po 14 p相互作用核酸酶。第三个具体目标是描述 5'羟基和3'磷酸末端DSB损伤的处理。一种新型 质粒转化试验将用于探讨其程度和机制 修复。该测定、体外生物化学测定和细胞对 化学诱变剂将评估ORF YMR 156 c是3'端的假设。 酵母多核苷酸激酶的磷酸酶部分。

DESCRIPTION (provided by applicant): Cancer is a leading cause of death and morbidity. It results from the accumulation of genetic mutations that ultimately lead to a growth advantage and expansion of rare cellular clones. Understanding the etiology of genetic mutation is thus of tremendous importance. Fundamentally, mutations represent the failure of a cell to correctly repair the DNA lesions caused by a variety of endogenous and exogenous damaging agents. This proposal is based on the model that inefficient repair of DNA double-strand breaks (DSBs) leads to persistence of lesions that ultimately become substrates for chromosomal rearrangement, a hallmark of malignancy. It focuses specifically on the enzymes that process DSB ends to make them ready for ligation, since failure of these enzymes might contribute substantially to persistence of DSB lesions in recombinogenic forms. All experiments use Saccharomyces cerevisiae as a model organism. State-of-the-art genomic tools are used to study many yeast genes in parallel and in combination, which is critical since multiplicity and redundancy of end processing pathways is anticipated. The first Specific Aim is to identify enzymes that resect the 5'-terminated strand in homologous recombinational repair (HRR). Preliminary evidence suggests that these cause an efficient and irreversible commitment to HRR in budding yeast. Novel competitive assays are based on the hypothesis that impaired 5' resection will increase the contribution of nonhomologous end-joining (NHEJ) and the likelihood of chromosomal rearrangement. High probability candidate helicases and nucleases will be examined in detail, in addition to panel and mutational screens for other involved genes. The second Specific Aim is to elucidate the mechanism of POL4 (yeast DNA polymerase b)-dependent processing in NHEJ. The hypothesis that this gene's previously described role represents an overlap with base excision repair is examined by chimeric analysis with the human enzyme. Protein interaction, panel and mutational screens will be used to identify Po14p-interacting nucleases. The third Specific Aim is to describe the processing of 5' hydroxyl and 3' phosphate terminal DSB lesions. A novel plasmid transformation assay will be used to explore the extent and mechanism of repair. This assay, in vitro biochemical assays, and cellular responses to chemical mutagens will evaluate the hypothesis that ORF YMR156c is the 3' phosphatase portion of yeast polynucleotide kinase.