MOLECULAR ANALYSIS OF SITE SPECIFIC DNA RECOMBINATION

位点特异性 DNA 重组的分子分析

• 批准号: 2179360
• 负责人: REID C JOHNSON
• 金额: $ 33.13万
• 依托单位: UNIVERSITY OF CALIFORNIA LOS ANGELES
• 依托单位国家: 美国
• 财政年份: 1987
• 资助国家: 美国
• 起止时间: 1987-07-01 至 1997-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2179360
• 关键词: DNA; DNA binding protein; DNA footprinting; Salmonella; X ray crystallography; bacterial proteins; binding proteins; crosslink; electron microscopy; enzyme mechanism; flagellin; gel electrophoresis; gene expression; gene mutation; gene rearrangement; genetic enhancer element; genetic manipulation; genetic mapping; genetic promoter element; genetic recombination; genetic regulation; immunoelectron microscopy; molecular cloning; nucleic acid sequence; nucleoproteins; protein biosynthesis; protein structure; recombinase; site directed mutagenesis; stoichiometry

Site-specific DNA recombination reactions control many types of biological reactions. These include many forms of gene regulation, viral integration and excision, chromosome partitioning and the combinatorial joining of gene segments to create new genes. While site-specific rearrangements can be part of the normal developmental program of specialized cells, aberrant rearrangements can lead to cancer and birth defects. The proposed work is concerned with understanding the molecular mechanisms involved in a model site-specific recombination reaction that regulates the alternative synthesis of flagellin proteins (flagellar phase variation) in Salmonella. This DNA inversion reaction can be studied in a purified in vitro system consisting of three proteins: Hin, Fis, and HU. The supercoiled DNA substrate must contain the two recombination sites plus a recombinational enhancer sequence, that together with the Fis protein, functions to increase the rate of inversion 102-103-fold in vivo and in vitro. Initiation of DNA strand exchange requires the formation of an invertasome structure in which the two recombination sites bound by the Hin recombinase are associated with the enhancer bound by Fis. Hin also has the ability to associate the two hix recombination sites into a complex, but without the enhancer, the paired-hix complex is incapable of supporting recombination. A variety of approaches will be used to discern the biochemical events that are required for invertasome assembly and strand exchange. We will attempt to determine if the paired-hix complex is a precursor to the invertasome and what molecular changes are triggered by the association of the enhancer. In order to do this, the protein interfaces that occur between and among Hin and Fis promoters in the different nucleoprotein complexes will be determined using mutational and site-directed crosslinking techniques. Some of these experiments will be guided by our initial mutation and crystal structure analysis of Fis as well as the published crystal structure of resolvase, which is related to Hin. Experiments are proposed which address the role of DNA supercoiling in invertasome assembly and recombination and to provide evidence that Hin-mediated recombination in vivo occurs in an invertasome structure. It has been proposed that DNA strand exchange is mediated by the rotation of Hin subunits in the invertasome complex. An attempt will be made to provide direct biochemical evidence for this mechanism. Both the Hin and Fis proteins specifically interact with DNA in novel ways. Fis binds to a highly degenerate DNA sequence and induces significant distortion of the DNA upon binding; stable Hin binding requires specific minor groove interactions. Intensive efforts to generate crystal structures of these proteins bound to DNA are ongoing. Mutagenesis and binding studies will also be used to specifically address Fis-DNA recognition and the nature and importance of Fis-induced DNA bending for enhancer function. The findings obtained from this work should be applicable to the many systems where the assembly of complex nucleoprotein complexes are required to promote various biological reactions.

位点特异性DNA重组反应控制许多类型的生物学过程。 反应. 其中包括多种形式的基因调控、病毒整合 切除、染色体分割和基因的组合连接 片段来创造新的基因 虽然特定地点的重新安排可以 特化细胞正常发育程序的一部分，异常的 基因重排可能导致癌症和先天缺陷。 拟议的工作是 关注于理解模型中的分子机制， 一种位点特异性重组反应， 沙门氏菌鞭毛蛋白质的合成（鞭毛相变异）。 这种DNA倒位反应可以在纯化的体外系统中进行研究 由三种蛋白质组成：Hin，Fis和HU。 超螺旋DNA 底物必须含有两个重组位点加上一个重组位点， 增强子序列与Fis蛋白一起发挥作用， 在体内和体外使转化率增加102-103倍。 DNA链交换的起始需要转化酶体的形成 Hin重组酶结合的两个重组位点 与Fis结合的增强子相关。 Hin也有能力 将两个hix重组位点结合成一个复合物，但没有 增强子，配对hix复合物不能支持重组。 将使用各种方法来辨别生化事件， 是转化体组装和链交换所必需的。 我们将尝试 以确定配对的hix复合物是否是转化体的前体 以及这些分子的结合会引发什么样的分子变化， 增强剂 为了做到这一点，蛋白质之间发生的界面， 不同核蛋白复合物中Hin和Fis启动子之间 将使用突变和定点交联来确定 技术. 其中一些实验将由我们最初的指导 突变和晶体结构分析以及已发表的 解离酶的晶体结构，与Hin有关。 实验 提出了解决DNA超螺旋在转化体组装中的作用 并提供证据表明Hin介导的重组 在体内发生在转化体结构中。 有人提出DNA 链交换是由Hin亚基在链中的旋转介导的。 倒置体复合体 将尝试提供直接的生物化学 这一机制的证据。 Hin和Fis蛋白都以新的方式与DNA特异性相互作用。 Fis与高度简并的DNA序列结合，并诱导显著的 结合时DNA的扭曲;稳定的Hin结合需要特异性的 小沟互动 加紧努力，以产生晶体 这些蛋白质与DNA结合的结构正在进行中。 诱变和 结合研究也将用于专门针对Fis-DNA 识别和性质和重要性的Fis-induced DNA弯曲， 增强子功能 从这项工作中获得的结果应该适用于许多 需要组装复杂核蛋白复合物的系统 以促进各种生物反应。