RECOMBINATION IN VITRO: ENZYMOLOGY AND INTERMEDIATES

体外重组：酶学和中间体

• 批准号: 3283300
• 负责人: CHARLES M. RADDING
• 金额: $ 38.08万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1984
• 资助国家: 美国
• 起止时间: 1984-05-01 至 1994-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3283300
• 关键词: DNA; DNA binding protein; DNA topoisomerases; affinity chromatography; bacterial genetics; circular DNA; conformation; enzyme complex; enzyme mechanism; enzyme structure; enzyme substrate; gel filtration chromatography; gene mutation; genetic manipulation; genetic recombination; nucleic acid sequence; nucleoproteins; polymerization; protein reconstitution; protein structure; radiotracer; recombinase; tissue /cell culture

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.

促进同源配对和链交换的酶学 由E.ColirecA蛋白质独一无二。鉴于复制和 转录涉及由大集合构建的多酶复合体 在不同的多肽链中，recA蛋白聚合在 单链DNA治疗多分子螺旋酶 能够促进同源配对和链的机器 交换。只有一种蛋白质，即大肠杆菌单链DNA 已知结合蛋白(SSB)直接参与recA 蛋白质在配对和链交换中，但其他酶必须 创造破坏的DNA底物，recA蛋白可以在上面作用 必须分解由recA蛋白产生的中间产物。《长河》 在这里提出的研究的术语目标是定义 从酶的角度看，构成主要途径的一连串事件 在大肠杆菌中进行同源重组。一种手段是实现这一目标，而且 一个中间目标是完全重建同源的 纯化的酶进行体外重组。我们的短程 目标是：1)建立和发展基于 用于灵敏检测的Lambda DNA的包装 大肠杆菌粗提物中DNA分子的重组，2) 通过使用特定结构的噬菌体lambda 在已知的大肠杆菌突变体中进行杂交，以阐明 可能是体内所需的一组酶 主要途径控制的同源重组 重组，recABCD途径，3)使用包装试验 (以上项目I和活体观察(以上项目2)至 开发一个从粗提物开始的体外系统。作为一名 纯化和鉴定所需的最小一组酶的手段 同源重组，4)研究基因突变的变化 纯化的全套已知重组活性的recA蛋白 从新的recA突变体中分析其致病机制 同源配对和链交换；5)进一步分析 拓扑要求，DNA构象的变化，以及 参与同源配对的中间体 带有螺旋recA核蛋白细丝的裸露双链DNA。 同源重组是遗传学的基石之一，是一种 具有根本利益和重要性的主题。对.的使用 特定染色体上“靶向”基因的同源重组 哺乳动物细胞中的位置可能会显著扩展遗传工具 可用于研究哺乳动物细胞，并可能最终提供一个 完成基因治疗所必需的手段。