MOLECULAR ANALYSIS OF SITE SPECIFIC DNA RECOMBINATION

许多细胞过程是由精确的相互作用控制的 在多种因素之间。我们才刚刚开始理解 多组分核蛋白复合体的分子水平 组装并催化活化。HIN位点特异性DNA 反转反应非常适合于研究这些问题。 在这个反应中，三个DNA片段和至少两个Hin 重组酶二聚体和两个FIS增强子蛋白二聚体组装 在需要DNA的反应中转化为转化体结构 超级卷曲。Invertasome组件也由 原核HU或真核HMG1/2家族成员 促进DNA环路的特定DNA结合蛋白。一个主要的 本项目的目的是阐明该分子的分子结构 催化能力强的转化体。结构信息是 适用于每个组件和几何图形 重组复合体内DNA链的构型是 为人所知。提出了一系列实验，这些实验是在 部分，通过计算机模拟来阐明其分子结构 转化体及其如何被FIS+催化激活 增强子DNA片段。此外，通过该机制 DNA链在转化体内交换将是 调查过了。这些研究将增进我们对 专业DNA重组反应的过程和控制， 它们存在于许多生物学环境中。异常重组 这些事件可能会导致严重的染色体异常和癌症。这个 非组蛋白DNA结合蛋白在多种反应中的作用 越来越多地被认为是 控制力。例如，FIS蛋白不仅调节Hin- 催化DNA倒置，但也控制转录 不同的启动子和刺激病毒从 染色体，另一种特定部位的DNA重组反应。这个 FIS激活和抑制转录和转录的机制 将研究lambda切除中的功能，特别是 确定FIS介导的DNA弯曲与 与适当的靶蛋白直接接触。这个 高亲和力结合位点与非特异性结合位点的分布 大肠杆菌基因组的不同区域也将被 探索提供有关FIS在 控制生长阶段和生长速度基因的表达和 调节整个类核结构。

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.