RECOMBINATION IN VITRO--ENZYMOLOGY AND INTERMEDIATES

体外重组——酶学和中间体

• 批准号: 6519144
• 负责人: CHARLES M. RADDING
• 金额: $ 52.54万
• 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 2004-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6519144
• 关键词: Escherichia coli; bacterial DNA; bacterial genetics; bacterial proteins; bacteriophage M13; chemical kinetics; deoxyribonuclease I; enzyme complex; enzyme mechanism; enzyme structure; enzyme substrate; gene targeting; genetic crossing over; genetic recombination; hydrogen bond; intermolecular interaction; nucleoproteins

E. Coli RecA protein forms a right-handed helical nucleoprotein filament on single-stranded DNA which catalyzes a search for homology, assimilates homologous duplex DNA, and effects the complete exchange of strands to produce a new heteroduplex recombinant. This protein appears to be the prototype of homologous recombination proteins that have recently been found to be universally distribute in nature, including Homo sapiens. The university of such proteins underscores the importance of understanding their molecular mechanisms, which may be important in disease processes that involve derangements of repair and recombination, and which will be of increasing interest in regard to gene targeting and gene therapy. The mechanisms of homologous pairing and strand exchange via the helical recombination filament are still not well understood, but intermediates have been identified and isolated that permit further enzymological and chemical probing. Recent studies have revealed that RecA protein can catalyze homologous recognition through the formation of non-Watson-Crick bonds. Experiments are proposed to probe the nature and role of these intermediates to test the hypothesis that homologous recognition is accomplished by a non-Watson-Crick interaction between a RecA coated single strand and duplex DNA. The rotation of DNA has been shown to be important in strand exchange. Experiments will test the hypothesis that rotation is accomplished by the coaxial rotation of DNA within the nucleoprotein filament. Experiments in vitro reveal the strand exchange phase of the recombination reaction only when a substrate is provided with an appropriate free end. A search will be made for enzymes that will cleave the initial synaptic complex made by RecA protein into one that can progress to the next stage of strand exchange. To this end, substrates and assays will be developed that can be used in crude extracts. Such methods may prove useful in the enzymolifocal approach to homologs of RecA protein in eukaryotes. The short term goals of the proposed research are to gain understanding of the mechanisms of homologous recognition and strand exchange and to discover how the intermediate that is generated by RecA protein is further processed. The long term goals are to understand the pathways of recombination mediated by RecA protein and its homologs, and to use that knowledge to facilitate gene targeting and gene therapy.

E. Coli RecA蛋白形成右手螺旋核蛋白丝 在单链DNA上催化寻找同源性， 同源双链DNA，并实现链的完全交换， 产生新的异源双链重组体。 这种蛋白质似乎是 同源重组蛋白的原型， 在自然界中广泛分布，包括智人。 这些蛋白质的大量存在强调了 了解它们的分子机制，这可能对 涉及修复和重组紊乱的疾病过程， 这将在基因靶向方面引起越来越多的兴趣， 基因治疗 同源配对和链交换的机制 通过螺旋复合丝的方式仍然没有很好的理解，但是 已经鉴定并分离出中间体， 酶学和化学探测。 最近的研究表明， RecA蛋白可以通过形成 非沃森-克里克债券 提出了实验来探索 和这些中间体的作用，以测试假设，同源 识别是通过一个非沃森-克里克之间的相互作用， RecA包被的单链和双链DNA。 DNA的旋转一直是 在链交换中显示出重要性。 实验将测试 假设旋转是由DNA的同轴旋转完成的 在核蛋白丝内。 体外实验表明， 重组反应的链交换阶段只有当底物 具有适当的自由端。 我们会搜查 这些酶将切割RecA产生的初始突触复合物， 将蛋白质转化为可以进行下一阶段链交换的蛋白质。 为此，将开发可用于以下的底物和测定： 粗提物 这些方法可能证明在酶促反应中是有用的。 RecA蛋白在真核生物中同源物的研究进展 短期目标 研究的目的是了解 同源识别和链交换，并发现 由RecA蛋白产生的中间体被进一步加工。 的 长期目标是了解重组介导的途径， 通过RecA蛋白及其同源物，并利用这些知识，以促进 基因靶向和基因治疗。