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Molecular analysis of genetic recombination and DNA break repair

基因重组和DNA断裂修复的分子分析

基本信息

批准号：
10681208
负责人：
GERALD R SMITH
金额：
$ 97.11万
依托单位：
FRED HUTCHINSON CANCER CENTER
依托单位国家：
美国
项目类别：
财政年份：
2016
资助国家：
美国
起止时间：
2016-05-01 至 2026-04-30
项目状态：
未结题

项目摘要

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.

项目总结/摘要这里提出的研究的长期目标是确定同源的分子机制，基因重组和DNA断裂修复。这一目标是通过遗传学的组合来实现的。突变体分析和来自细胞的蛋白质和DNA的生化分析。这项研究利用裂变酵母裂殖酵母以及大肠杆菌及其噬菌体λ。都是被广泛研究的高度易处理的模式生物，具有包括人类在内的所有生物的共同特征。本研究主要集中在S.粟酒，其高重组率促进遗传和生化分析，以及重组和DNA断裂修复的主要途径，细菌，由RecBCD酶促进，RecBCD酶是一种复杂的DNA修复机器，其3D结构使我们能够在原子水平上确定重组起始是如何被调节的。在过去成就的基础上，目前的研究重点是以下领域。1)研究减数分裂DNA双链断裂（DSB）热点如何形成簇，以及这些簇如何赋予DSB 干扰，因此，交叉干扰对于正确的染色体分离是重要的。这研究有望解决100年来的交叉干扰问题，这是一个主要的遗传难题，我们已经提出了一个分子机制，并得到了许多数据的支持。2)研究RecBCD如何酶控制其潜在的猖獗的核酸酶活性，并通过与Chi相互作用适当地激活它。重组热点（5 ′ GCTGGTGG 3 ′）。这项研究有望在近原子水平上解决 RecBCD酶是E.杆菌重组，首次观察到75年前，和一个范例的染色体位点控制的其他复杂 DNA酶。3)寻找更有效的RecBCD抑制剂，这是一种有前途的抗生素，（未使用）目标。需要新的抗生素来对抗越来越频繁的耐药细菌。这些目标将通过突变体的遗传分析，细胞内荧光显微镜蛋白质和染色体位点，来自减数分裂细胞的DNA中间体的物理分析，以及酶和分离的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断裂修复以及控制重组，确保它发生在在染色体上的正确时间和位置减数分裂染色体的分离是重要的，经常无错误的修复，引起DNA双链断裂，以及细胞和生物多样性的产生。畸变重组可产生染色体重排，如易位、复制和缺失，这些疾病通常与不孕症、出生缺陷和癌症有关，或者是它们的原因。