MOLECULAR MECHANISMS OF GENETIC RECOMBINATION

这里提出的研究的长期目标是确定 从酶的角度探讨广义遗传重组的机制 促进这一进程的各个步骤和 每一步都有DNA中间体。另一个目标是确定 重组被调控的机制。我们建议开发 一种源自大肠杆菌的无细胞重组系统。我们的 重组的底物将是来自噬菌体Lambda衍生物的DNA 可能在反向重复序列之间进行分子内重组。 在大肠杆菌提取物中孵育后，这种DNA将被 体外包装成可用选择性平板法检测的噬菌体 亲本和重组型。我们的初步调查和那些 证明在受感染的细胞中，这种重组是由 转化需要确定的大肠杆菌重组酶-RecA和 RECBC-并且受到CHI站点的刺激。我们将确定要求 对于额外的蛋白质，如DNA连接酶和单链结合 蛋白质(SSB)，以及其他过程，如DNA复制。与 无手机系统，我们将确定任何其他所需的组件和 确定DNA中间体的结构。我们将继续我们的 大肠杆菌RecBC酶的研究以确定其作用机制 它可以将DNA解开和倒带。我们将确定DNA的RecBC，以及它的 在其他蛋白质存在的情况下，解离和重绕的速率，例如 RecA和SSB，已知参与重组。通过对……的研究 我们将研究recBC的分子遗传学调控 RecBC基因的表达，绘制了recBC的精细结构图谱 基因，继续与我们初步分离的Lambda recBC克隆一起工作 工作，并寻找recBC突变体以评估recBC的生理作用 酶及其调节其合成和活性的机制。

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 propose to develop a cell-free system for recombination derived from Escherichia coli. Our substrate for recombination will be DNA from a derivative of phage Lambda that may undergo intramolecular recombination between inverted repeats. Following incubation in an E. coli extract, this DNA will be converted by in vitro packaging into phage which can be assayed by selective plating for parental and recombinant types. Our preliminary investigations and those of others demonstrate that in infected cells this recombination-mediated inversion requires identified E. coli recombinational enzymes - RecA and RecBC - and is stimulated by Chi sites. We will determine the requirement for additional proteins, such as DNA ligase and single-strand binding protein (SSB), and for other processes, such as DNA replication. With the cell-free system, we will identify any additional required components and determine the structures of the DNA intermediates. We will continue our studies of the RecBC enzyme of E. coli to determine the mechanism by which it unwinds and rewinds DNA. We will determine RecBC's of DNA, and its rates of unwinding and rewinding in the presence of other proteins, such as RecA and SSB, known to be involved in recombination. Through a study of the molecular genetics of recBC we will investigate the regulation of expression of the recBC genes, develop a fine structure map of the recBC genes, continue work with Lambda recBC clones isolated in our preliminary work, and seek recBC mutants to evaluate the physiological roles of RecBC enzyme and the mechanisms that regulate its synthesis and activity.