Molecular Analysis of Hotspots of Genetic Recombination

描述（由申请人提供）：拟开展的长期研究目标是阐明同源基因重组和DNA断裂修复的分子机制。这一目标是通过研究重组热点来实现的，这激发了重组的一个关键的、限速的步骤。在大肠杆菌中，研究将集中在Chi热点，它刺激重组和DNA断裂修复的主要途径（RecBCD）。在分裂酵母Schizosaccharomyces pombe中，研究将集中在突变产生的M26热点和mbs1等自然产生的热点上。这些微生物特别适合进行遗传和生化分析，但在许多方面，它们的重组模仿了人类的重组。具体目的是：1)阐明Chi热点与RecBCD酶的复杂相互作用，特别强调测试Chi触发的RecBCD亚基间信号传导的特定假设；2)确定关节分子形成和分解所需的蛋白质，包括新的单（而不是双）Holliday连接，并在这方面比较不同的热点。3)确定染色质修饰和染色体蛋白在调控整个基因组热点中的作用。这些目标将通过结合生物化学和纯化组分的电子显微镜，以及完整细胞的遗传学和DNA分析来实现。这些研究结果将阐明重组的机制及其在染色体和生物体生命周期中的调控。重组在染色体中DNA双链断裂的忠实修复和减数分裂中染色体的忠实分离中起着重要的作用。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明显原因。RecBCD及其密切相关的酶广泛分布于细菌中，而不是真核生物中，因此可能是一类急需的新型抗生素的良好靶点。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：基因重组对DNA断裂的修复和减数分裂（性细胞形成）期间染色体的适当分离很重要。错误的重组会导致癌细胞或先天缺陷。通过研究重组的热点，我们将扩大对这一对人类健康至关重要的过程的分子基础的认识。

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.