BINDING OF E COLI LEXA REPRESSOR TO DNA

大肠杆菌 LEXA 阻遏物与 DNA 的结合

• 批准号: 2185293
• 负责人: SANDRA L SHANER
• 金额: $ 10.36万
• 依托单位: WAYNE STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1992
• 资助国家: 美国
• 起止时间: 1992-08-01 至 1997-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2185293
• 关键词: DNA footprinting; Escherichia coli; acidity /alkalinity; affinity chromatography; bacterial DNA; bacterial genetics; bacterial proteins; biophysics; chemical association; chemical binding; chemical kinetics; fluorescence spectrometry; gel mobility shift assay; genetic operator element; ionic strengths; polymerization; regulatory gene; solutions; stoichiometry; temperature; thermodynamics; transcription factor

The long-term objective of this research program is to understand the regulation of the SOS response in E. coli. The SOS response is an inducible network of functions promoting DNA repair and cellular survival which is activated when E. coli is treated with agents that damage cellular DNA or block its replication. Inducible responses to DNA damage exist in a number of other bacteria and apparently in a variety of eucarytic systems as well. Genetic and biochemical data suggest that activation of the SOS response results upon derepression of a class of genes following the specific proteolytic cleavage of their common repressor, the LexA repressor, by RecA protein. At least 17 different genes, necessary for functions as diverse as mutagenesis, DNA repair, recombination, cell division and prophage induction, have been identified as under LexA repressor control. In particular, this research program aims to understand the physical forces that determine the ability of LexA repressor to recognize and to bind to its operators in the promoter regions of the various genes within this regulon. Such information should increase understanding of the general energetic principles that may determine specificity of DNA recognition in other protein-nucleic acid binding systems. Recognition of specific DNA sequences by proteins is involved in the control of many cellular processes (eg, replication, transcription and translation). Therefore, in addition to understanding the mechanism by which E. coli copes with DNA damage, such studies are potentially relevant to understanding the molecular origin of and developing therapeutic strategies for control of diseases involving defects in any of these fundamental cellular processes. The specific experimental goals for this grant period are: 1) to investigate the thermodynamics of the nonspecific binding interactions of LexA repressor with single-stranded and double-stranded DNA. Equilibrium binding constants will be characterized as a function of the concentrations and type of electrolyte ions, pH and temperature; 2) to investigate the thermodynamics of the site-specific interaction of LexA repressor with the reca and uvrA operator sites on duplex DNA restriction fragments as a function of solution conditions; 3) to investigate the dependence of the self-association reaction of LexA repressor on solution conditions parallel to those used in the studies of the equilibrium binding of LexA repressor to DNA; 4) to initiate kinetic studies of the interaction of LexA repressor with the recA operator, 5) to initiate thermodynamic studies of the equilibrium binding of LexA repressor to the dual operator sites located at the regulatory region of the lexa gene using quantitative DNA footprinting.

该研究计划的长期目标是了解 大肠杆菌中SOS反应的调节。 SOS响应是 促进DNA修复和细胞存活的功能网络的诱导网络 当大肠杆菌用损伤细胞处理的药物处理时，它会被激活 DNA或阻止其复制。 对DNA损伤的诱导反应存在 其他细菌的数量，显然是在各种桉树系统中 也是如此。 遗传和生化数据表明SOS的激活 响应结果是在遵循一类基因的压迫之后的结果 其共同阻遏物的特定蛋白水解切割lexa 抑制剂，通过RECA蛋白。 至少17种不同的基因，需要 与诱变，DNA修复，重组，细胞一样多样化 划分和预言归纳已被确定为Lexa 阻遏物控制。 特别是，该研究计划旨在了解 确定Lexa阻遏物能力的物理力 认识并与其在发起人区域的运营商结合 该调节中的各种基因。 这样的信息应增加 了解可能决定的一般能量原则 在其他蛋白核酸结合中DNA识别的特异性 系统。 通过蛋白质识别特定的DNA序列参与 控制许多细胞过程（例如，复制，转录和 翻译）。 因此，除了了解机制 大肠杆菌应对DNA损伤，此类研究可能是相关的 了解和发展治疗的分子起源 控制涉及缺陷的疾病的控制策略 基本的细胞过程。为此的具体实验目标 赠款期是：1）调查非特异性的热力学 Lexa阻遏物与单链和 双链DNA。 平衡结合常数将被表征 作为电解质离子的浓度和类型的函数，pH和 温度; 2）研究特定地点的热力学 Lexa阻遏物与RECA和UVRA操作员站点的相互作用 双链DNA限制片段与溶液条件的关系； 3） 研究Lexa的自我关联反应的依赖性 在解决方案条件下与研究中使用的溶液条件的阻遏物 Lexa抑制剂与DNA的平衡结合； 4）发起动力学 Lexa阻遏物与RECA操作员的相互作用的研究，5） 启动Lexa阻遏物平衡结合的热力学研究 到位于Lexa监管区域的双操作员站点 使用定量DNA足迹的基因。