Molecular Analysis of Site Specific DNA Recombination

本计画的长期目标是了解核蛋白的组装、控制与功能 促进Hin催化的位点特异性DNA倒位的复合物。上一个供资期间的工作 建立了Hin突触复合物的四聚体催化核心的整体结构，提供了强的 证据表明，DNA链的重组是由Hin亚基对在细胞内的旋转介导的。 四聚体，并显示了Fis/增强子系统如何与DNA超螺旋一起控制亚基旋转。 未来的工作将需要一个整体的遗传，生物化学和结构的方法，并将强调 Fis/增强子元件激活初始反应步骤以及控制 亚基旋转过程机理分析将从不同类别的丝氨酸的酶开始 重组酶，强调其新颖的特性。该项目的一个突出主题是DNA结合 类核蛋白在重组和转录反应中的性质和调节作用。新 将继续研究通过类核蛋白控制噬菌体λ位点特异性重组的方面。X-的 类核蛋白Fis结合的复合物的X射线晶体结构分析将扩展到更高- 有序复合物，其含有噬菌体λ Xis蛋白和RNA的C-末端结构域 聚合酶α亚基（alphaCTD）。这些结构研究将提供新的信息， DNA构象柔性对蛋白质间接识别和协同DNA结合的影响。细胞Fis 在生长阶段和生长速率方面， E.大肠杆菌在快速生长条件下，但几乎没有在稳定期。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机制。 最近的体外研究表明，Fis可能在调节染色体结构中发挥重要作用 因为它的DNA弯曲和循环活动。Fis对染色体结构的影响将得到解决 通过体内和体外方法的组合，包括协同的单DNA分子方法， 以及通过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.