Molecular Analysis of Site Specific DNA Recombination

描述(由申请人提供)：该项目的一个持续的长期目标是了解促进Hin催化的位点特异性DNA反转的核蛋白复合体的组装、控制和功能。过去资助期的工作确立了Hin突触复合体四聚体催化核心的总体结构，并提供了强有力的证据，表明DNA链的重组是通过在二聚体之间转移Hin亚单位来介导的。未来的工作将需要一系列的遗传、生化和结构方法，旨在获得发生在突触上的结构变化的更高分辨率的观点，以及激活DNA催化和交换的机制(S)。辅助染色质蛋白的关键但不同的作用已经成为这项工作的第二个突出主题。FIS(倒置刺激因子)被发现是因为它在Hin和Gin DNA倒置反应中起着重要的调节作用，但现在已知它在大量的DNA反应中起作用。我们将研究FIS如何与噬菌体lambda的Xis蛋白合作来刺激噬菌体切除，最终目标是详细了解控制lambda位点特异性重组方向性的结合协作性和DNA结构变化。细胞FIS水平随着生长阶段和生长速度的不同而变化很大：在快速生长条件下，FIS是大肠杆菌中含量最丰富的转录调节因子，而在稳定期几乎不存在。我们将分析FIS在不同生长条件下对基因表达和蛋白质水平的全基因组影响，并从分子上剖析潜在的新调控单位。FIS通过与RNA聚合酶α亚单位(AlphaCTD)的C末端区域特异性地相互作用来激活转录。将追踪Fis-DNA和Fis-AlphaCTD-DNA络合物的X射线晶体结构。除了在特定的DNA结合部位形成稳定的复合体外，FIS还在生理浓度下与DNA非特异性相互作用，并可能由于其DNA弯曲和环化活性而在调节染色体结构中发挥重要作用。FIS对染色体结构的影响将通过单DNA分子方法以及体相体外和体内实验来共同解决。

DESCRIPTION (provided by applicant): A continuing 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 and provided strong evidence that recombination of DNA strands was mediated by translocating Hin subunits between dimers. Future work will entail an ensemble of genetic, biochemical, and structural approaches that are directed at obtaining higher resolution views of the structural changes that occur upon synapsis and the mechanism(s) by which Fis-activates DNA catalysis and exchange. The crucial yet varied roles of accessory chromatin proteins has emerged as a second prominent theme of this work. Fis (Factor for Inversion Stimulation) was discovered because of its essential regulatory role in the Hin and Gin DNA inversion reactions, but it is now known to function in a large number of DNA reactions. We will investigate how Fis collaborates with the Xis protein of phage lambda to stimulate phage excision, with the ultimate goal of obtaining a detailed understanding of binding cooperativity and DNA structural changes that control the directionality of lambda site-specific recombination. Cellular Fis levels vary enormously with respect to growth phase and growth rates: Fis is the most abundant transcriptional regulator in E. coli under rapid growth conditions, whereas it is virtually absent in stationary phase. We will profile genome-wide effects of Fis on gene expression and protein levels under different growth conditions and molecularly dissect potentially novel regulatory units. Fis activates transcription by specifically interacting with the C-terminal domain of the RNA polymerase alpha subunit (alphaCTD). X-ray crystal structures of Fis-DNA and Fis-alphaCTD-DNA complexes will be pursued. In addition to forming stable complexes at specific DNA binding sites, Fis also interacts nonspecifically with DNA at physiological concentrations and may play important roles in modulating chromosome structure because of its DNA-bending and looping activities. Fis effects on chromosome structure will be collaboratively addressed by single DNA molecule approaches as well as by bulk-phase in vitro and in vivo experiments.