MOLECULAR ANALYSIS OF SITE-SPECIFIC DNA RECOMBINATION

定点DNA重组反应控制着多种类型的生物 反应。其中包括多种形式的基因调控、病毒整合 以及切除、染色体分割和基因的组合连接 来创造新的基因。而特定于场地的重新安排可以是 特化细胞正常发育程序的一部分，异常 重排可能会导致癌症和出生缺陷。建议的工作是 与理解模型中涉及的分子机制有关的 调节选择性的位点特异性重组反应 沙门氏菌鞭毛蛋白的合成(鞭毛相变异)。 这种DNA反转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 底物必须包含两个重组位点和一个重组 增强子序列，与FIS蛋白一起，起到 将体内和体外的反转率提高102-103倍。 DNA链交换的启动需要形成转化体 由HIN重组酶结合的两个重组位点的结构 与FIS结合的增强子相关。Hin也有能力 将两个HIX重组位点关联到一个复合体中，但没有 增强子，配对-HIX复合体不能支持重组。 将使用各种方法来识别 是进行转化体组装和链交换所必需的。我们将尝试 为了确定配对的HIX复合体是否是转化体的前体 以及哪些分子变化是由分子间的关联触发的 增强剂。为了做到这一点，发生在 不同核蛋白复合体中HIN和FIS启动子之间的差异 将使用突变和定点定向交联法来确定 技巧。其中一些实验将由我们最初的 FIS的突变和晶体结构分析以及已发表的 分解酶的晶体结构，这与Hin有关。实验是 提出了DNA超螺旋在转化体组装中的作用 和重组，并提供Hin介导的重组的证据 在体内发生在倒体体结构中。有人提出，DNA 链交换是通过Hin亚基的旋转来调节的 转化体复合体。将尝试提供直接的生化反应 这一机制的证据。 Hin和Fis蛋白都以新的方式与DNA特异地相互作用。 FIS与高度简并的DNA序列结合并诱导显著的 DNA结合时的扭曲；稳定的HIN结合需要特定的 次要凹槽相互作用。密集努力生产水晶 这些蛋白质与DNA结合的结构正在进行中。诱变和 结合研究也将被用来专门处理FIS-DNA 对FIS诱导的DNA弯曲的认识及其性质和重要性 增强功能。 从这项工作中获得的发现应该适用于许多人 需要组装复杂核蛋白复合体的系统 促进各种生物反应。

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.