RECOMBINATION IN VITRO--ENZYMOLOGY AND INTERMEDIATES

E.Coli RecA蛋白形成右旋螺旋核蛋白细丝 关于催化同源、同化搜索的单链DNA 同源双链DNA，并影响链的完全交换 产生一种新的异源双链重组。这种蛋白质似乎是 最近发现的同源重组蛋白的原型 发现在自然界中普遍分布，包括智人。 这类蛋白质的大学强调了 了解它们的分子机制，这可能对 涉及修复和重组错乱的疾病过程， 这将会引起人们对基因靶向和 基因疗法。同源配对和链交换的机制 通过螺旋复合细丝，仍然不是很清楚，但 中间体已被鉴定和分离，允许进一步 酶学和化学探查。最近的研究表明， RecA蛋白可以催化同源识别通过形成 非沃森-克里克债券。有人提议用实验来探索这一性质 以及这些中间体的作用来检验同源的假说 识别是通过一个非Watson-Crick交互作用实现的 RecA包被的单链和双链DNA。DNA的旋转已经被 在链交换中被证明是重要的。实验将测试 假设旋转是通过DNA的同轴旋转来完成的 在核蛋白细丝内。体外实验揭示了 只有当底物为底物时，链交换阶段的复合反应才会发生 具有适当的自由端。我们将会寻找 裂解RecA形成的初始突触复合体的酶 蛋白质转化为一种可以进行下一阶段链交换的蛋白质。 为此，将开发可用于 粗提物。这些方法可能在酶免疫定位中被证明是有用的。 真核生物中RecA蛋白同源物的研究。短期目标 这项拟议的研究的目的是为了了解 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。这个 长期目标是了解重组介导的途径 通过RecA蛋白及其同源物，并利用这一知识来促进 基因打靶和基因治疗。

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.