MOLECULAR MECHANISMS OF GENETIC RECOMBINATION

本研究的长期目标是 确定广义遗传重组的机制， 促进这一过程的各个步骤的酶的术语 过程和DNA中间体的结构在每个步骤。 另一个目标是确定 重组是有规律的。 我们将继续研究 大肠杆菌RecBCD酶，这是所需的 重组的主要途径。杆菌 我们将确定 这种酶解开和重绕DNA的机制 并水解DNA 我们将结合以下活动： RecBCD酶和RecA和SSB蛋白，以产生 双链体DNA底物的重组中间体。 我们 将决定RecBCD酶切割D环的能力 并解决霍利迪路口，两个假定的中间体， 重组 我们将分离并研究突变体 改变RecBCD酶的多种活性，以阐明 它们在重组和其他细胞过程（恢复）中的作用 从DNA损伤和维持细胞活力）。 我们将 我们继续研究recBCD的表达调控 基因，以阐明一种机制，重组可能是 控制。 为了比较生物体中重组的机制， 比E. coli中，我们将克隆编码RecBCD样的基因， 其他细菌的酶，并确定这些酶的能力， 促进重组的酶。大肠杆菌中， 细菌 我们将研究这些酶的活性， DNA. 我们将寻找重组相关的酶 在裂殖酵母裂殖酵母中的活性。 我们 将分离S。粟酒裂殖酵母突变体在重组中改变或缺乏 相关酶活性。 这些突变体和酶将 为研究其分子机制提供了基础。 真核生物中的重组，并比较这些机制 与原核生物中的那些相同。 生物多样性是生物多样性的重要组成部分， 有机体和细胞水平。 重组异常 产生染色体重排，如缺失， 易位 染色体重排与 可能是先天缺陷和癌症的原因。 理解重组的分子机制是 重要的是确定的原因，并可能防止 这些疾病。

The long-term objective of the research proposed here is to determine the mechanism of generalized genetic recombination in terms of the enzymes promoting the individual steps of this process and the structures of the DNA intermediates at each step. An additional objective is to determine the mechanisms by which recombination is regulated. We will continue our studies of the Escherichia coli RecBCD enzyme, which is required for the principal pathway of recombination in E. coli. We will determine the mechanisms by which this enzyme unwinds and rewinds DNA and by which it hydrolyzes DNA. We will couple the activities of RecBCD enzyme and RecA and SSB proteins to produce recombinational intermediates from duplex DNA substrates. We will determine the ability of RecBCD enzyme to cleave D-loops and to resolve Holliday junctions, two postulated intermediates in recombination. We will isolate and study mutants differentially altered in the multiple activities of RecBCD enzyme to elucidate their roles in recombination and other cellular processes (recovery from DNA damage and maintenance of cell viability). We will continue our studies of the regulation of expression of the recBCD genes, to elucidate a mechanism by which recombination may be controlled. To compare the mechanisms of recombination in organisms other than E. coli, we will clone the genes coding for RecBCD-like enzymes of other bacteria and determine the ability of these enzymes to promote recombination in E. coli and in the original bacteria. We will investigate the activities of these enzymes on DNA. We will search for recombination-related enzymatic activities in the fission yeast Schizosaccharomyces pombe. We will isolate S. pombe mutants altered in recombination or lacking relevant enzymatic activities. These mutants and enzymes will provide a foundation for studying the molecular mechanisms of recombination in eukaryotes and for comparing these mechanisms with those in prokaryotes. Recombination is important for generating diversity at both the organismal and cellular levels. Aberrancies of recombination generate chromosomal rearrangements, such as deletions and translocations. Chromosomal rearrangements are associated with, and may be a cause of, birth defects and cancers. Understanding the molecular mechanisms of recombination is important in determining the causes of and possibly preventing these diseases.