Molecular Analysis of Site Specific DNA Recombination

描述（由申请人提供）：本项目的长期目标是了解促进hin催化位点特异性DNA倒置的核蛋白复合物的组装、控制和功能。过去资助期间的工作建立了Hin突触复合物的四聚体催化核心的整体结构，并提供了强有力的证据，证明DNA链的重组是通过在二聚体之间转移Hin亚基介导的。未来的工作将需要遗传学、生物化学和结构方法的整合，以获得突触发生的结构变化的更高分辨率视图，以及fiss激活DNA催化和交换的机制。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 （Factor for Inversion Stimulation，倒置刺激因子）因其在Hin和Gin DNA倒置反应中具有重要的调控作用而被发现，但现在已知它在大量DNA反应中起作用。我们将研究Fis如何与噬菌体lambda的Xis蛋白协同作用以刺激噬菌体切除，最终目的是详细了解结合协同性和控制lambda位点特异性重组方向性的DNA结构变化。细胞中Fis的水平随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是大肠杆菌中最丰富的转录调节因子，而在固定生长条件下，Fis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，并对潜在的新调控单元进行分子解剖。Fis通过特异性地与RNA聚合酶α亚基（alphaCTD）的c端结构域相互作用来激活转录。fish - dna和fish - alphactd - dna复合物的x射线晶体结构将被研究。除了在特定的DNA结合位点形成稳定的复合物外，Fis还在生理浓度下与DNA非特异性相互作用，并且由于其DNA弯曲和环化活性，可能在调节染色体结构中发挥重要作用。它对染色体结构的影响将通过单DNA分子方法以及体外和体内的大量实验来共同解决。

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.