Molecular Analysis of Hotspots of Genetic Recombination

描述（由申请人提供）：拟议研究的长期目标是阐明同源遗传重组和DNA断裂修复的分子机制。这一目标是通过研究热点的重组，刺激一个关键的，限速的重组步骤。在大肠杆菌中，研究将集中在Chi热点，它刺激重组和DNA断裂修复的主要（RecBCD）途径。在裂殖酵母裂殖酵母中，研究将集中在突变产生的M26热点和mbs1和其他自然发生的热点上。这些微生物特别适合于遗传和生化分析，但在许多方面，它们的重组模拟了人类的重组。具体目标是1）阐明Chi热点和RecBCD酶的复杂相互作用，特别强调测试由Chi触发的RecBCD亚基间信号传导的特定假设，2）确定形成和解析联合分子所需的蛋白质，包括新的单克隆抗体。（而不是双）霍利迪连接，并在这方面比较不同的热点，以及3）确定染色质修饰和染色体蛋白在整个基因组中调节热点的作用。这些目标将通过生物化学和电子显微镜与纯化组分的组合以及遗传学和完整细胞的DNA分析来实现。这些研究的结果将阐明重组的机制及其沿沿着染色体和在生物体的生命周期中的调节。 在染色体DNA双链断裂的忠实修复和减数分裂过程中染色体的忠实分离中，脱附是重要的。 重组和DNA断裂修复的异常是与癌症、出生缺陷和某些遗传性疾病相关的染色体畸变的原因。RecBCD和密切相关的酶广泛分布在细菌中，但不是真核生物，因此可能是一类急需的新抗生素的良好靶标。因此，这里提出的基础研究将为理解、诊断、预防和治疗人类疾病奠定基础。公共卫生相关性：遗传重组对于修复断裂的DNA和在减数分裂（性细胞形成）期间染色体的正确分离是重要的。基因重组可导致癌细胞或出生缺陷。通过研究重组的热点，我们将扩大我们对这一过程的分子基础的知识，这对人类健康很重要。

DESCRIPTION (provided by applicant): The long-term goal of the proposed research is to elucidate the molecular mechanism of homologous genetic recombination and DNA break repair. This goal is approached by studying hotspots of recombination, which stimulate a critical, rate-limiting step of recombination. In the bacterium Escherichia coli, studies will focus on Chi hotspots, which stimulate the major (RecBCD) pathway of recombination and DNA break repair. In the fission yeast Schizosaccharomyces pombe, studies will focus on the mutationally created M26 hotspot and on mbs1 and other naturally occurring hotspots. These microbes are especially amenable for genetic and biochemical analyses, but in many ways their recombination mimics that of humans. The specific aims are 1) to elucidate the complex interaction of Chi hotspots and RecBCD enzyme, with special emphasis on testing a specific hypothesis of RecBCD inter-subunit signaling triggered by Chi, 2) to determine the proteins required for formation and resolution of joint molecules, including novel single (rather than double) Holliday junctions, and to compare different hotspots in this respect, and 3) to determine the roles of chromatin modifications and chromosomal proteins in regulating hotspots throughout the genome. These aims will be achieved by a combination of biochemistry and electron microscopy with purified components, and genetics and DNA analysis with intact cells. The results of these studies will elucidate both the mechanism of recombination and its regulation along chromosomes and during the organism's life cycle. Recombination is important in the faithful repair of DNA double-strand breaks in chromosomes and in the faithful segregation of chromosomes during meiosis. Aberrancies of recombination and DNA break repair are responsible for chromosomal aberrations associated with and apparent causes of cancer, birth defects, and certain hereditary diseases. RecBCD and closely related enzymes are widely distributed among bacteria but not eukaryotes and may therefore be good targets for a new class of critically needed antibiotics. Thus, the basic research proposed here will add to the foundations for understanding, diagnosing, preventing, and curing human disease. PUBLIC HEALTH RELEVANCE: Genetic recombination is important for the repair of broken DNA and for the proper segregation of chromosomes during meiosis (sex-cell formation). Faulty recombination can result in cancer cells or birth defects. By studying hotspots of recombination we shall expand our knowledge of the molecular basis of this process important for human health.