MOLECULAR MECHANISMS OF GENETIC RECOMBINATION

本文提出的研究的长期目标是确定 广义遗传重组的酶机制 促进这一进程的各个步骤和 每一步的DNA中间体。 另一个目标是确定 调节重组的机制。 我们建议发展 源自大肠杆菌的用于重组的无细胞系统。 我们 重组底物是来自噬菌体λ衍生物的DNA 可以在反向重复序列之间进行分子内重组。 在E.大肠杆菌提取物，这种DNA将被转化为 体外包装入噬菌体，其可以通过选择性平板接种来测定， 亲本和重组类型。 我们的初步调查和那些 其他研究表明，在受感染的细胞中， 转化需要鉴定E.大肠杆菌重组酶- RecA和 RecBC -并受到Chi站点的刺激。 我们将确定要求 对于其他蛋白质，如DNA连接酶和单链结合 蛋白质（SSB）和其他过程，如DNA复制。 与 在无细胞系统中，我们将确定任何额外的所需组件， 确定DNA中间体的结构。 我们将继续 对E.大肠杆菌，以确定其机制， 它会解开并倒回DNA 我们将确定DNA的RecBC， 在其他蛋白质存在下的解旋和重绕速率，例如 RecA和SSB，已知参与重组。 通过研究 我们将研究recBC的分子遗传学， recBC基因的表达，开发recBC的精细结构图， 基因，继续与我们的初步分离的Lambda recBC克隆工作 工作，并寻求recBC突变体，以评估RecBC的生理作用 酶和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.