Molecular Analysis of Site Specific DNA Recombination

这个项目的一个长期目标是了解核蛋白的组装、控制和功能。 促进Hin催化的位点特异性DNA倒置的复合体。过去资助期内的工作 建立了四聚体催化核心的Hin突触复合体的整体结构，提供了强大的 有证据表明DNA链的重组是由Hin亚基对在 四聚体，并展示了FIS/增强子系统与DNA超螺旋一起控制亚单位旋转的方式。 未来的工作将涉及一系列遗传、生化和结构方法，并将强调 FIS/增强子元件激活初始反应步骤以及控制 亚基轮换过程。将对不同类别丝氨酸的一种酶进行机械分析 重组酶，强调其新奇的性质。该项目的一个突出主题是DNA绑定 类核蛋白的性质及其在重组和转录反应中的调节作用。新的 在控制噬菌体Lambda位点特异性重组方面，将继续进行类核蛋白的研究。X- 类核蛋白FIS结合的络合物的射线晶体结构分析将扩展到更高层次. 含有噬菌体lambda Xis蛋白和RNA的C-末端结构域的有序复合体 聚合酶α亚基(αCTD)。这些结构研究将提供有关以下方面的新信息： 蛋白质对DNA的间接识别和协同结合的构象灵活性。蜂窝FIS 在不同的生长阶段和生长速度下，DNA含量差异很大，是最丰富的DNA 在快速生长条件下的大肠杆菌中的结合蛋白，但在静止时几乎不存在。机制 将研究FIS通过特异性和非特异性DNA结合进行转录调控的情况。 最近的体外证据表明，fis可能在调节染色体结构方面发挥重要作用。 因为它的DNA弯曲和环路活动。FIS对染色体结构的影响将被解决 通过体内和体外相结合的方法，包括协作单DNA分子方法 以及体相体外和体内实验。

A long term goal of this project is to understand the assembly, control, and function of nucleoprotein complexes that promote Hin-catalyzed site-specific DNA inversion. Work during the past funding period established the overall structure of the tetrameric catalytic core of the Hin synaptic complex, provided strong evidence that recombination of DNA strands was mediated by rotation of Hin subunit pairs within the tetramer, and showed how the Fis/enhancer system together with DNA supercoiling controls subunit rotation. Future work will entail an ensemble of genetic, biochemical, and structural approaches and will emphasize the mechanism by which the Fis/enhancer element activates initial reaction steps as well as controls the subunit rotation process. Mechanistic analysis will commence on an enzyme from a different class of serine recombinase, emphasizing its novel properties. A prominent theme of the project concerns the DNA binding properties and regulatory roles of nucleoid proteins on recombination and transcription reactions. New aspect on the control of phage lambda site specific recombination by nucleoid proteins will be pursued. X- ray crystal structure analyses of complexes bound by the nucleoid protein Fis will be extended to higher- order complexes which contain the phage lambda Xis protein and the C-terminal domain of the RNA polymerase alpha subunit (alphaCTD). These structural studies will provide new information on the role of DNA conformational flexibility on indirect recognition and cooperative DNA binding by proteins. Cellular Fis levels vary enormously with respect to growth phase and growth rates, being the most abundant DNA binding protein in E. coli under rapid growth conditions but virtually absent in stationary phase. Mechanisms of transcriptional regulation by Fis through specific and non-specific DNA binding will be investigated. Recent in vitro evidence suggests Fis may play an important role in modulating chromosome structure because of its DNA-bending and looping activities. Fis effects on chromosome structure will be addressed by a combination of in vivo and in vitro approaches, including collaborative single-DNA molecule approaches as well as by bulk-phase in vitro and in vivo experiments.