MOLECULAR ANALYSIS OF SITE SPECIFIC DNA RECOMBINATION

许多细胞过程是由一个精确的相互作用控制的 多种因素之间的关系。我们才刚刚开始了解 多组分核蛋白复合物在分子水平上 组装并催化活化。Hin位点特异性DNA 转化反应非常适合研究这些问题。 在该反应中，三个DNA片段和至少2个Hin 重组酶二聚体和两个Fis增强子蛋白二聚体组装 转化成转化酶体结构 超螺旋倒置体组装也由以下任一者辅助： 原核HU或真核HMG 1/2家族成员， 促进DNA成环的特异性DNA结合蛋白。主 该项目的目的是阐明 催化活性转化体。 结构信息 可用于每个组件和几何 重组复合物内DNA链的构型是 知道的一系列的实验提出了指导，在 部分，通过计算机建模来阐明分子结构， 转化体及其如何被Fis plus催化活化 增强子DNA片段此外， DNA链在转化体内交换， 研究了这些研究将促进我们对 专门的DNA重组反应的过程和控制， 在许多生物学背景下都能找到。异常重组 事件可能导致严重的染色体畸变和癌症。的 非组蛋白DNA结合蛋白在不同反应中的功能 越来越多地被认为是一个重要的水平， 控制作为一个例子，Fis蛋白不仅调节Hin- 催化DNA倒位，但也控制转录， 不同的启动子和刺激病毒切除从 染色体，另一个位点特异性DNA重组反应。的 Fis激活和抑制转录的机制， 将研究lambda切除中的函数，特别是 确定Fis介导的DNA弯曲与 直接接触合适的靶蛋白。的 高亲和力位点与非特异性结合的分布 大肠杆菌基因组的不同区域也将被 探讨提供有关金融机构在以下方面的作用的信息： 控制生长期和生长速率基因表达， 介导整个类核结构。

Many cellular processes are controlled by a precise interplay between multiple factors. We are just beginning to understand at the molecular level how multi-component nucleoprotein complexes are assembled and catalytically activated. The Hin site-specific DNA inversion reaction is well suited for investigating these issues. In this reaction, three DNA segments and a minimum of 2 Hin recombinase dimers and two Fis enhancer protein dimers assemble into an invertasome structure in a reaction that requires DNA supercoiling. Invertasome assembly is also assisted by either the prokaryotic HU or members of the eukaryotic HMG1/2 family of non- specific DNA binding proteins that promote DNA looping. A primary aim of this project is to elucidate the molecular structure of the catalytically competent invertasome. Structural information is available for each of the components and the geometric configuration of DNA strands within the recombination complex is known. A series of experiments are proposed which are guided, in part, by computer modeling to elucidate the molecular structure of the invertasome and how it is catalytically activated by Fis plus the enhancer DNA segment. In addition, the mechanism by which the DNA strands are exchanged within the invertasome will be investigated. These studies will advance our understanding of the process and control of specialized DNA recombination reactions, which are found in many biological contexts. Aberrant recombination events can lead to serious chromosomal aberrations and cancer. The functions of non-histone DNA binding proteins in diverse reactions is becoming increasingly recognized as an important level of control. As an example, the Fis protein not only regulates Hin- catalyzed DNA inversion, but also controls transcription of different promoters and stimulates viral excision from the chromosome, another site-specific DNA recombination reaction. The mechanisms by which Fis activates and represses transcription and functions in lambda excision will be investigated, particularly to determine the relevant roles of Fis-mediated DNA bending versus direct contact with the appropriate target protein. The distribution of high affinity sites versus non-specific binding at different regions of the Escherichia coli genome will also be explored to provide information regarding the role of Fis in controlling growth phase and growth rate gene expression and in mediating overall nucleoid structure.