RECOMBINATION IN VITRO: ENZYMOLOGY AND INTERMEDIATES

同源配对和链交换的酶学促进了 由E. coli recA蛋白的独特性。 而复制和 转录涉及从大集合构建的多酶复合物 recA蛋白聚合在 单链DNA处理多分子和螺旋酶 能够促进同源配对和链的机器 交易所 只有一种蛋白质，E。大肠杆菌单链DNA 已知结合蛋白（SSB）直接参与recA 蛋白质的配对和链交换，但其他酶必须 产生recA蛋白可以作用的断裂DNA底物， 必须分解recA蛋白产生的中间产物。 长 本文提出的研究目标是确定 从酶的角度看， 在E.杆菌 一种是达到这一目的的手段， 一个中间目标是完全重建同源的 从纯化的酶体外重组。 我们的短程 目标是：1）建立和开发基于 包装λ DNA，以便灵敏地检测 重组E.大肠杆菌，2） 通过使用噬菌体λ的特定构建体， 在E.大肠杆菌，以阐明 可能是体内所需的一组酶， 同源重组的主要途径控制 重组，recABCD途径，3）使用包装测定 (item I和体内观察结果（上文第2项）， 从粗提物开始开发体外系统。 作为 是指纯化和鉴定所需的最小酶集， 同源重组，4）研究突变的变化， 纯化的recA蛋白的整套已知重组活性 从新的recA突变体中，以分析 同源配对和链交换，以及5）进一步分析 拓扑要求，DNA构象的变化， 参与同源配对的中间体 裸双链DNA与螺旋recA核蛋白丝。 同源重组是遗传学的基石之一， 这是一个重要的、具有根本意义的问题。 使用 同源重组将基因“靶向”到特定染色体 哺乳动物细胞中的位点可能会显著扩展遗传工具， 可以用来研究哺乳动物细胞，并可能最终提供一个 完成基因治疗的必要手段。

The enzymology of homologous pairing and strand exchange promoted by E. coli recA protein's unique. Whereas replication and transcription involve multi-enzyme complexes built from large sets of different polypeptide chains, recA protein polymerizes on single-stranded DNA to treat a multimolecular and helical enzymatic machine that is capable of promoting homologous pairing and strand exchange. Only one other protein, E. coli single-stranded DNA binding protein (SSB) is known to participate directly with recA protein in pairing and strand exchange, but other enzymes must create the broken DNA substrates on which recA protein can act, and must resolve the intermediates created by recA protein. The long term objective of the research proposed here is to define enzymatically the chain of events that constitute the major pathway of homologous recombination in E. coli. One means to that end, and an intermediate goal is the complete reconstitution of homologous recombination in vitro from purified enzymes. Our short range goals are: 1) to set up and develop the assay based on the packaging of lambda DNA in order to detect sensitively recombination of DNA molecules in crude extracts of E. coli, 2) through the use of particular constructs of phage lambda for crosses in known mutants of E. coli, to elucidate as far as possible the set of enzymes that are required in vivo for homologous recombination controlled by the principal pathway of recombination, the recABCD pathway, 3) to use the packaging assay (item I above and the in vivo observations (item 2 above) to develop an in vitro system starting from crude extracts. as a means to purify and identify a minimal set of enzymes required for homologous recombination, 4) to study mutational changes in the entire set of known recombine activities of recA protein purified from new recA mutants in order to analyze the mechanism of homologous pairing and strand exchange, and 5) to analyze further the topological requirements, the changes in DNA conformation, and the intermediates that are involved in the homologous pairing of naked duplex DNA with the helical recA nucleoprotein filament. Homologous recombination, one of the keystones of genetics, is a subject of fundamental interest and importance. The use of homologous recombination to "target" genes to specific chromosomal sites in mammalian cells may significantly expand the genetic tools available to study mammalian cells and may eventually provide one of the means necessary to accomplish gene therapy.