RECOMBINATION IN VITRO: ENZYMOLOGY AND INTERMEDIATES

E.大肠杆菌recA蛋白在体外促进同源配对 单链或部分单链DNA以三种顺序 步骤：1）recA蛋白在单链 2）突触，DNA的连接和同源排列 分子，和3）链交换。 三种核蛋白结构具有 被鉴定为假定的中间体：1）突触前复合物， 其上聚合有recA蛋白的单链DNA，2）连接的 分子，双链DNA结合到突触前复合体， 同源配对，和3）突触或新生异源双链结构， 新的三链中间体，其中引入的链不 拓扑上与它的互补体相互缠绕，但尽管如此， 通过新生异源双链体连接。 通过酶学和生物化学 方法，我们将探索这些中间体的结构， 特别是在定义DNA链的接触点， recA蛋白。 我们研究了reCA蛋白与 用E.大肠杆菌：单链结合蛋白，T4基因32 蛋白质、拓扑异构酶I、DNA连接酶、recBC DNA酶、核酸外切酶I和λ 核酸外切酶 通过一系列涉及各种DNA底物的模型系统， 以及各种酶的组合，我们建议开发重组 适用于粗提取物或其级分的测定 为了在体外重建重组， 鉴定同源的主要途径的所有酶 重组在E.杆菌 为此目的，recA蛋白提供了一种 仪器试剂，因为它既把DNA分子在一起， 它们是同源排列的。 一方面， 重组尚未阐明;另一方面，研究 近年来通过重组DNA方法可能揭示了 重组不仅在遗传上而且在生物学上重要性 规范与发展。

E. coli recA protein in vitro promotes homologous pairing of single-stranded or partially single-stranded DNA in three sequential steps: 1) presynaptic polymerization of recA protein on single-stranded DNA, 2) synapsis, the conjunction and homologous alignment of DNA molecules, and 3) strand exchange. Three nucleoprotein structures have been identified as putative intermediates: 1) presynaptic complexes, single-stranded DNA with recA protein polymerized on it, 2) conjoined molecules, double-stranded DNA bound to presynaptic complexes without homologous pairing, and 3) synaptic or nascent heteroduplex structures, novel three-stranded intermediates in which the incoming strand is not topologically interwound with its complement but is nonetheless base-paired via a nascent heteroduplex joint. By enzymological and biochemical methods, we will explore the structures of these intermediates, aiming specifically at defining the points of contact of the strands of DNA and recA protein. We have studied the interactions of reCA protein with the following enzymes from E. coli: single-strand binding protein, T4 gene 32 protein, topoisomerase I, DNA ligase, recBC DNase, exonuclease I and Lambda exonuclease. By a series of model systems involving various DNA substrates and various combinations of enzymens we propose to develop recombination assays that are suitable for use with crude extracts or fractions thereof in order to reconstitute recombination in vitro and thereby to enable the identification of all of the enzymens of the major pathway of homologous recombination in E. coli. For this purpose, recA protein provides an instrumental reagent, since it both brings DNA molecules together and puts them in homologous alignment. On the one hand no single pathway of recombination has yet been elucidated; on the other hand, research made possible in recent years by recombinant DNA methodology has revealed the importance of recombination not only in inheritance but also in biological regulation and development.