End Processing in DNA Double Strand Break Repair

简介(申请人提供)：癌症是导致死亡的主要原因， 发病率。它是由于基因突变的积累造成的 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学具有巨大的意义 重要性。从根本上说，突变代表细胞不能 正确修复多种内源性和外源性激素引起的DNA损伤 外源性损害剂。这项建议是基于效率低下的模式 DNA双链断裂(DSB)的修复导致病变的持久性 最终成为染色体重排的底物，这是 恶毒。它特别关注将DSB末端加工成的酶 使它们准备好连接，因为这些酶的失败可能会导致 实质上与重组形式的DSB病变的持久性有关。全 实验以酿酒酵母为模式生物。最先进的 基因组学工具被用来同时研究许多酵母基因。 组合，这一点至关重要，因为终端的多样性和冗余性 加工路径是预期的。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，这些导致了一种有效和 在萌芽酵母中对HRR的不可逆转承诺。新的竞争性分析是 基于受损的5‘端切除会增加 非同源末端连接(NHEJ)的贡献及其可能性 染色体重排。高概率候选解旋酶和核酸酶 将详细检查，除了面板和突变筛查 其他涉及的基因。第二个具体目的是阐明其发病机制。 在NHEJ中依赖于POL4(酵母DNA聚合酶b)的加工。假设 该基因先前描述的作用与碱基切除有重叠 修复是通过与人类酶的嵌合分析进行检查的。蛋白 将使用交互、面板和突变屏幕来识别 Po14p相互作用的核酸酶。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。一本小说 通过质粒转化实验，对转化的程度和机制进行了探讨 修理的问题。这项检测、体外生化检测和细胞对 化学诱变剂将评估ORF YMR156c是3‘的假设 酵母菌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.