ENZYMATIC EXCISION AND REPAIR MECHANISMS

酶促切除和修复机制

• 批准号: 3484426
• 负责人: LAWRENCE GROSSMAN
• 金额: $ 26.35万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1978
• 资助国家: 美国
• 起止时间: 1978-09-01 至 1994-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3484426
• 关键词: DNA binding protein; DNA repair; Escherichia coli; adenosinetriphosphatase; bacterial DNA; bacterial genetics; bacterial proteins; chemical binding; chemical structure function; conformation; endonuclease; enzyme mechanism; exoribonucleases; gene deletion mutation; genetic manipulation; genetic mapping; genetic regulation; immunoelectroadsorption; nucleic acid sequence; nucleoproteins; oligonucleotides; peptidases; protein engineering; protein sequence; tissue /cell culture; zinc

The Escherichia coli uvr ABC system catalyzes the incision of damaged DNA. Having available reagent quantities of homogeneous UurB and UvrC proteins permits a detailed examination of the individual partial reactions leading to the dual incision stage of nucleotide excision repair. The pre-incision steps can be subdivided into a number of partial reactions which include: (a) UvrA dimerization stimulated by ATP binding, (b) UvrA-nucleoprotein formation at both damaged and undamaged sites, (c) the accompanying topological unwinding stimulated by ATP binding, (d) the participation of the UvrB cryptic ATPase in the UvrAB catalyzed strand displacement reaction and finally (e) dual incision catalyzed by the presence of UvrC. The incision mechanisms precede the multi-nucleoprotein complex requiring coordinated excision reactions catalyzed by UvrD, DNA polymerase I and polynucleotide ligase. In the principal direction for the proposed studies we will attempt to associate the anatomy, or structure of the respective uvr A and B genes to the catalytic and protein properties of the related gene product proteins. The focal points and role of ATP in the individual processes will also be addressed. The protein sequences, or domains, of interest include putative ATP binding regions, sites sensitive to a protease specific for the Ada protein of E. coli potential DNA binding sites and to a limited extent the "zinc finger"-like sites. These sites will be engineered by oligonucleotide-directed and deletion mutants, the individual clones sequenced and over-expressed in suitable expression vectors and the catalytic and protein properties of the mutant and "wild type" proteins examined for their enzymatic phenotypes in the respective pre-, post- and incision steps. The biological role and biochemical nature of UvrB proteolysis in regulation of repair will be further investigated by genetic, structural and catalytic methods.

大肠杆菌uvr ABC系统催化损伤的 DNA. 具有可用试剂量的均质UurB和UvrC 蛋白质允许详细检查单个部分 导致核苷酸切除双切口阶段的反应 修复. 切割前步骤可以细分为多个 部分反应包括：（a）由ATP刺激的UvrA二聚化 结合，（B）在受损和未受损处的UvrA-核蛋白形成 位点，（c）ATP刺激的伴随拓扑解旋 结合，（d）UvrB隐蔽ATP酶参与UvrAB 催化的链置换反应和最后（e）双切口 在紫外线C的催化下 切口机制先于 需要协同切除反应的多核蛋白复合物 由UvrD、DNA聚合酶I和多核苷酸连接酶催化。 在拟议研究的主要方向上，我们将努力 将uvr A和B基因各自的解剖学或结构相关联 相关基因产物的催化和蛋白质性质 proteins. ATP在各个过程中的协调中心和作用 也将得到解决。 感兴趣的蛋白质序列或结构域 包括推定ATP结合区、对蛋白酶敏感的位点 对大肠杆菌Ada蛋白具有特异性。coli潜在的DNA结合位点， 在有限程度上的“锌指”样位点。 这些网站将 通过寡核苷酸定向突变体和缺失突变体工程化， 对单个克隆进行测序并以合适的表达过量表达 载体以及突变体和“野生型”的催化和蛋白质性质 在相应的细胞中检查其酶表型的“型”蛋白质 术前、术后和切开步骤。 UvrB蛋白酶解的生物学作用和生化性质 修复的调节将通过遗传、结构、 和催化方法。