RECOMBINATION IN VITRO: ENZYMOLOGY AND INTERMEDIATES

大肠杆菌recA蛋白体外促进同源配对 单链或部分单链DNA分三个序列 步骤：1)单链RecA蛋白的突触前聚合 DNA，2)突触，DNA的连接和同源排列 分子，以及3)链交换。有三种核蛋白结构 被确定为假定的中间产物：1)突触前复合体， 聚合了recA蛋白的单链DNA，2)连接 分子，双链DNA结合到突触前复合体而没有 同源配对，以及3)突触或新生异源双链结构， 新型三链中间体，其中传入链不是 在拓扑上与其补语交织在一起，但仍然是碱基配对的 通过一个新生的异质双工关节。通过酶学和生化 方法，我们将探索这些中间体的结构，目的是 特别是在定义DNA链的接触点和 RecA蛋白。我们研究了RecA蛋白与细胞的相互作用。 来自大肠杆菌的以下酶：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Lambda 核酸外切酶。通过一系列涉及不同DNA底物的模型系统 和各种酶的组合，我们建议发展重组 适合与粗提物或其部分一起使用的分析 为了在体外重建重组，从而使 同源异构体主要途径的所有酶的鉴定 在大肠杆菌中重组。为此，recA蛋白提供了一种 仪器试剂，因为它既能将DNA分子聚集在一起，又能使 它们在同源排列中。一方面，没有单一的途径 重组尚未阐明；另一方面，研究发现 近年来通过重组DNA方法学的可能揭示了 重组的重要性不仅在遗传上，而且在生物学上也是如此 规范和发展。

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.