Molecular Analysis of Site Specific DNA Recombination

位点特异性 DNA 重组的分子分析

• 批准号: 7897421
• 负责人: REID C JOHNSON
• 金额: $ 21.63万
• 依托单位: UNIVERSITY OF CALIFORNIA LOS ANGELES
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-08-17 至 2010-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7897421
• 关键词: Address; Area; Bacteriophage lambda; Bacteriophages; Binding; Binding Sites; Biochemical; Biological; C-terminal; Catalysis; Catalytic Domain; Cell physiology; Chromosome Pairing; Chromosome Structures; Complex; DNA; DNA Binding; Equilibrium; Escherichia coli; Excision; Factor For Inversion Stimulation Protein; Funding; Future; Gene Expression; Gene Expression Regulation; Genes; Genetic; Genetic Recombination; Genetic Transcription; Goals; Grant; Growth; HMGB Proteins; In Vitro; Integrase; Lead; Measurement; Mediating; Molecular Analysis; Nucleoproteins; Peptides; Phase; Physiological; Play; Property; Proteins; RNA polymerase alpha subunit; Reaction; Regulation; Research; Resolution; Roentgen Rays; Role; Rotation; Series; Site; Solutions; Structure; Synapses; Work; chromatin protein; dimer; genome-wide; in vivo; mutant; novel; rapid growth; research study; single molecule

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.

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倒位的核蛋白复合物的组装、控制和功能。在过去的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表明，DNA链的重组是通过在二聚体之间易位Hin亚基介导的。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，旨在获得更高的分辨率视图的结构变化，发生在突触和机制（S），其中Fis-activates DNA催化和交换。辅助染色质蛋白质的重要而多样的作用已经成为这项工作的第二个突出主题。Fis（Factor for Inversion Stimulation）是由于其在Hin和Gin DNA反转反应中的重要调节作用而被发现的，但现在已知其在大量DNA反应中起作用。我们将研究Fis如何与λ噬菌体的Xis蛋白合作来刺激噬菌体切除，最终目标是详细了解控制λ位点特异性重组的方向性的结合协同性和DNA结构变化。细胞Fis水平随生长期和生长速率的不同而变化很大：Fis是大肠杆菌中最丰富的转录调节因子。大肠杆菌中，在快速生长条件下，而它几乎是不存在的稳定期。我们将在不同的生长条件下分析Fis对基因表达和蛋白质水平的全基因组效应，并从分子水平上剖析潜在的新型调控单位。Fis通过与RNA聚合酶α亚基（alphaCTD）的C末端结构域特异性相互作用来激活转录。将追求Fis-DNA和Fis-alphaCTD-DNA复合物的X射线晶体结构。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弯曲和成环活性而可能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。