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ENZYMATIC EXCISION AND REPAIR MECHANISMS

酶促切除和修复机制

基本信息

批准号：
2770895
负责人：
LAWRENCE GROSSMAN
金额：
$ 40.32万
依托单位：
JOHNS HOPKINS UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
1978
资助国家：
美国
起止时间：
1978-09-01 至 2000-08-31
项目状态：
已结题

项目摘要

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.

大肠埃希氏菌UVR ABC系统催化损伤组织的切割 DNA有均质UurB和UvrC的有效试剂量蛋白质允许对个体部分进行详细检查导致核苷酸切除双切口期的反应修理。切开前的步骤可以细分为多个步骤部分反应包括：(A)三磷酸腺苷刺激的UvrA二聚化结合，(B)UvrA-核蛋白在受损和未受损时形成站点，(C)伴随的由三磷酸腺苷刺激的拓扑解除结合，(D)UvrB隐蔽ATPase参与UvrAB 催化链置换反应和最终(E)双重切割由UvrC的存在催化。切开机构先于需要协同切除反应的多核蛋白复合体由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，我们将尝试将各自的UVR A和B基因的解剖或结构联系起来对相关基因产物的催化和蛋白质性质的影响蛋白质。ATP在个体过程中的焦点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、对蛋白酶敏感的位置针对大肠杆菌潜在DNA结合部位的Ada蛋白和在有限的程度上出现了“锌指”样的网站。这些网站将是由寡核苷酸导向和缺失突变体设计的对单个克隆进行测序，并在适当的表达中过度表达突变体和野生型载体及其催化和蛋白质性质类型“的蛋白质在各自的前、后、切三步。 UvrB蛋白水解酶的生物学作用和生化性质修复的调节将进一步从遗传、结构和催化法。