Molecular analysis of genetic recombination and DNA break repair

项目摘要/摘要 本研究的长期目标是确定同源基因的分子机制。 基因重组和DNA断裂修复。这一目标是通过基因的组合来实现的 突变分析和细胞中蛋白质和DNA的生化分析。这项研究使用了裂变 酵母菌、酵母菌、大肠杆菌及其噬菌体。都是 被广泛研究的、高度易驯化的模式生物，具有包括人类在内的所有生物的共同特征。 这些研究主要集中在减数分裂重组，其高的重组率有利于 遗传和生化分析以及重组和DNA断裂修复的主要途径 细菌，由RecBCD酶促进，一种复杂的DNA修复机器，其3D结构允许我们 在原子水平上确定重组启动是如何被调控的。 在以往研究成果的基础上，目前的研究主要集中在以下几个方面。1)学习 减数分裂DNA双链断裂(DSB)热点如何形成簇，以及这些簇如何传递DSB 因此，交叉干扰对于正确的染色体分离很重要。这 研究承诺解决100年来的交叉干扰问题，这是人类主要的遗传难题 我们提出了一种分子机制，并得到了大量数据的支持。2)研究RecBCD如何 酶控制其潜在的核酸酶活性，并通过与CHI的相互作用适当地激活它 重组热点(5‘GCTGGTGG3’)。这项研究承诺在近原子水平上解决 大肠杆菌主要途径的主要控制者RecBCD酶的分子机制 75年前首次观察到的重组，以及其他复合体的染色体位置控制范例 DNA酶。3)寻找更有效的RecBCD抑制剂，这些抑制剂有望对抗一种新的 (未使用)目标。需要新的抗生素来对抗日益频繁的抗药性细菌。这些 目标将受到突变的遗传分析、细胞内荧光显微镜的组合攻击 蛋白质和染色体位置，减数分裂细胞DNA中间体的物理分析，以及酶和 分离蛋白质的生物物理分析。这些研究的结果将阐明分子机制。 重组和DNA断裂修复以及对重组的控制，以确保它发生在 染色体上适当的时间和地点。 重组对于忠实的减数分裂染色体分离、频繁的无错误修复是重要的 引起DNA双链断裂，并产生细胞和生物多样性。* 重组可以产生染色体重排，如易位、复制和缺失， 它们通常与不孕不育、出生缺陷和癌症有关或导致不孕、出生缺陷和癌症。

Project Summary/Abstract The long-term goal of the research proposed here is to determine the molecular mechanism of homologous genetic recombination and DNA break repair. This objective is approached by a combination of genetic analysis of mutants and biochemical analysis of proteins and DNA from cells. This research uses the fission yeast Schizosaccharomyces pombe as well as the bacterium Escherichia coli and its phage lambda. All are widely studied, highly tractable model organisms with features common to all organisms, including humans. The studies are focused on meiotic recombination in S. pombe, whose high rates of recombination facilitate both genetic and biochemical analyses, and on the major pathway of recombination and DNA break repair in bacteria, promoted by RecBCD enzyme, a complex DNA repair machine, whose 3D structure allows us to determine at atomic level how recombination initiation is regulated. Building on past achievements, the research is currently focused on the following areas. 1) Studying how meiotic DNA double-strand break (DSB) hotspots form clusters, and how these clusters impart DSB interference and, consequently, crossover interference important for proper chromosome segregation. This research promises to solve the 100-year-old problem of crossover interference, a major genetic puzzle for which we have proposed a molecular mechanism and supported with many data. 2) Studying how RecBCD enzyme controls its potentially rampant nuclease activity and appropriately activates it by interaction with Chi hotspots of recombination (5’ GCTGGTGG 3’). This research promises to solve at near-atomic level the molecular mechanism of RecBCD enzyme, the principal controller of the major pathway of E. coli recombination, first observed 75 years ago, and a paradigm for chromosomal site control of other complex DNA enzymes. 3) Seeking more potent RecBCD inhibitors, which are promising antibiotics against a novel (unused) target. New antibiotics are needed to counter ever-more-frequent drug-resistant bacteria. These goals will be attacked by a combination of genetic analysis of mutants, fluorescence microscopy of intracellular proteins and chromosomal sites, physical analysis of DNA intermediates from meiotic cells, and enzymatic and biophysical analyses of isolated proteins. The results of these studies will elucidate the molecular mechanism of recombination and DNA break repair as well as the controls on recombination that ensure that it occurs at the proper time and place along chromosomes. Recombination is important for faithful meiotic chromosome segregation, error-free repair of frequently arising DNA double-strand breaks, and generation of cellular and organismal diversity. Aberrancies of recombination can generate chromosomal rearrangements, such as translocations, duplications, and deletions, which are often associated with or the cause of infertility, birth defects, and cancers.