T4 ENDONUCLEASE V--STRUCTURE/FUNCTION ANALYSES

T4 核酸内切酶 V--结构/功能分析

• 批准号: 2153558
• 负责人: R. Stephen Lloyd
• 金额: $ 22.15万
• 依托单位: UNIVERSITY OF TEXAS MED BR GALVESTON
• 依托单位国家: 美国
• 财政年份: 1992
• 资助国家: 美国
• 起止时间: 1992-07-01 至 2000-09-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2153558
• 关键词: DNA repair; N glycosidase; X ray crystallography; active sites; chemical binding; chemical cleavage; chemical kinetics; computer simulation; conformation; crosslink; endonuclease; enzyme mechanism; enzyme structure; enzyme substrate; enzyme substrate complex; fluorescent dye /probe; molecular cloning; mutant; nuclear magnetic resonance spectroscopy; nucleic acid structure; protein sequence; pyrimidine dimers; scanning electron microscopy; site directed mutagenesis; transmission electron microscopy

There is a well established link between inefficient DNA repair of ultraviolet (UV) light induced damage and carcinogenesis in man, as demonstrated through the study of the human disease, xeroderma pigmentosum and epidemiological studies of nonmelanoma skin cancer. Enhanced UV exposure and persistence of dipyrimidine photoproducts have been correlated with oncogene activation and tumor suppressor inactivation . Thus, the ability to maintain DNA integrity is rooted in accurate DNA repair systems that catalyze the restoration of damaged DNAs to their native state as well as high fidelity DNA replication. The variety of ubiquitous DNA repair pathways include recombinational, nucleotide, and base excision repair. The initial steps of the base excision pathway consist of the location, recognition, and release of damaged bases by DNA glycosylases. These enzymes sometimes have a concomitant AP lyase activity. One of these enzymes, T4 endonuclease V, is a cyclobutane pyrimidine dimer DNA glycosylase-AP lyase. Previous studies have revealed the fundamental mechanisms by which this enzyme initiates repair at this UV-induced lesion, and include the determination of its active site, the sequences that are responsible for DNA binding and the chemical basis for catalysis. In order for endonuclease V to continue to serve as the prototype for mechanistic studies of this class of enzymes, the following specific aims are proposed: 1) Develop fine structure kinetic analyses of endonuclease V-catalyzed reactions. order to determine the progression of steps that ultimately lead to enzyme-mediated catalysis, a full kinetic heme will be developed using rapid quench-flow technologies and novel DNA substrates. 2) Determine the molecular architecture of endonuclease V bound to cyclobutane dimer.containing DNA. It is hypothesized that endonuclease V undergoes a conformational change on dimer-specific binding. This hypothesis will be tested by biophysically characterizing DNA-protein completes before and after the glycosylase step. 3) Describe the architecture of endonuclease V-nontarget DNA interactions and determine the mechanism of target site location. A variety of genetic and biophysical methods will be used to investigate hypotheses concerning the associations between nontarget DNA and endonuclease V. 4) Determine the analytic mechanisms for other pyrimidine dimer-specific DNA glycosylases-AP lyases. It is hypothesized that all DNA glycosylases-AP lyases function through a common chemistry. This will be tested by elucidating the catalytic mechanism of action for other dimer-specific DNA glycosylase-AP lyases. These studies may the foundation for developing unifying principles for initiating the restoration of damaged DNA by DNA glycosylases. In the long term, this will involve the following determinations: 1) what are the mechanisms by se proteins locate sites of specific damage within vast excesses of undamaged DNA; 2) what are the principles for specific substrate recognition and what are the structural motifs within these proteins that achieve differential binding; 3) what are the active site residues within these enzymes and how do these residues suggest catalytic mechanisms; 4 what is the chemical basis for glycosyl bond cleavage?

在效率低下的DNA修复之间存在良好的联系 紫外线（UV）的光引起人类的损害和致癌作用，因为 通过研究人类疾病的研究证明 非甲状腺瘤皮肤癌的流行病学研究。增强的紫外线 二吡二酰亚胺光产物的暴露和持久性已相关 癌基因激活和肿瘤抑制灭活。因此， 保持DNA完整性的能力植根于精确的DNA修复系统 这也将损坏的DNA恢复到其祖国状态 作为高保真DNA复制。多种无处不在的DNA修复 途径包括重组，核苷酸和碱基切除修复。这 基本切除途径的初始步骤由位置组成， DNA糖基酶识别并释放受损碱。这些 酶有时具有伴随的AP溶酶活性。其中之一 酶，T4核酸内切酶V，是环丁烷嘧啶二聚体DNA 糖基酶-ap裂解酶。先前的研究表明了基本 该酶在此紫外线诱导的病变处启动修复的机制， 并包括确定其活性位点的确定 负责DNA结合和催化的化学基础。为了 核酸内切酶V继续充当机械的原型 对这类酶的研究，提出了以下特定目的： 1）开发核酸内切酶V催化的精细结构动力学分析 反应。为了确定最终领导的步骤的进展 为了酶介导的催化，将开发完整的动力学血红素 快速淬灭流技术和新型DNA底物。 2）确定 核酸内切酶V与环丁烷结合的分子结构 二聚体。假设核酸内切酶V发生A 二聚体特异性结合的构象变化。这个假设将是 通过生物物理表征DNA-蛋白质的测试 糖基酶步骤之后。 3）描述核酸内切酶的结构 V-nontarget DNA相互作用并确定目标位点的机理 地点。多种遗传和生物物理方法将用于 研究有关非目标DNA之间关联的假设 4）确定其他其他的分析机制 嘧啶二聚体特异性DNA糖基酶-AP裂解酶。它是假设的 所有DNA糖基酶-AP裂解酶通过常见的化学作用。 这将通过阐明作用的催化机理来测试 其他二聚体特异性的DNA糖基酶-AP裂解酶。这些研究可能是 制定统一原则的基础 通过DNA糖基酶恢复受损的DNA。从长远来看，这将 涉及以下确定：1）SE的机制是什么 蛋白质在巨大过度损坏的地方找到特定损害的部位 脱氧核糖核酸; 2）特定底物识别的原则是什么？ 是这些蛋白质中的结构基序 结合； 3）这些酶中的活性位点残基是什么？ 这些残基是否提示催化机制？ 4什么是化学物质 糖基键裂解的基础？