BINDING OF E COLI LEXA REPRESSOR TO DNA

大肠杆菌 LEXA 阻遏物与 DNA 的结合

基本信息

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
项目类别：
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

项目摘要

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 修复和细胞存活的诱导功能网络当大肠杆菌被破坏细胞的物质处理时，它就会被激活 DNA或阻止其复制。对 DNA 损伤的诱导反应存在于许多其他细菌，显然在各种真核系统中以及。遗传和生化数据表明 SOS 的激活反应结果是一类基因去抑制后的结果其共同阻遏物 LexA 的特异性蛋白水解切割抑制子，由 RecA 蛋白产生。至少需要 17 个不同的基因功能多样，如诱变、DNA 修复、重组、细胞分裂和前噬菌体诱导，已根据 LexA 进行鉴定抑制器控制。特别是，该研究计划旨在了解决定 LexA 阻遏物能力的物理力识别并结合到其启动子区域的操纵子该调节子内的各种基因。此类信息应增加了解可能决定的一般能量原理其他蛋白质-核酸结合中 DNA 识别的特异性系统。蛋白质对特定 DNA 序列的识别涉及许多细胞过程的控制（例如复制、转录和翻译）。因此，除了了解其机制外，大肠杆菌应对 DNA 损伤，此类研究具有潜在相关性了解分子起源并开发治疗方法控制涉及这些缺陷的疾病的策略基本的细胞过程。本次具体实验目标资助期限为： 1）研究非特异性的热力学 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浓度和类型、pH 和温度; 2）研究特定位点的热力学 LexA 阻遏物与 reca 和 uvrA 操作位点的相互作用双链 DNA 限制性片段作为溶液条件的函数； 3）研究 LexA 自缔合反应的依赖性溶液条件上的阻遏物与研究中使用的条件平行 LexA阻遏物与DNA的平衡结合； 4) 启动动力 LexA 阻遏物与 recA 操纵子相互作用的研究，5) 启动 LexA 阻遏物平衡结合的热力学研究位于 lexa 监管区域的双运营商站点基因使用定量 DNA 足迹法。