MOLECULAR EFFECTORS OF ENZYMATIC SYNTHESIS OF DNA

DNA 酶促合成的分子效应器

• 批准号: 6385616
• 负责人: MUKUND J MODAK
• 金额: $ 27.48万
• 依托单位: UNIV OF MED/DENT OF NJ-NJ MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 1985
• 资助国家: 美国
• 起止时间: 1985-08-05 至 2004-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6385616
• 关键词: DNA directed DNA polymerase; DNA replication; affinity labeling; enzyme mechanism; enzyme model; enzyme structure; enzyme substrate; gene mutation; nucleic acid biosynthesis; nucleic acid sequence; site directed mutagenesis

DESCRIPTION (adapted from applicant's abstract): The enzymatic synthesis of DNA is a multistep process that requires sequential binding of substrate, accompanied by several conformational changes within the enzyme protein. The molecular mechanisms involved in these steps are not well understood. The recent availability of a number of crystal structures of this class of enzymes, however, has made a significant advancement whereby the basic molecular mechanisms of DNA polymerization and their relationship to the structural makeup of enzyme may be clarified at the atomic level. The major objective of this proposal is to continue investigations on the biochemical, enzymological and structural properties of the prototype enzyme, namely E. coli polymerase 1. The choice of this enzyme in the proposed study is based on the fact that a) the three dimensional anatomy of large fragment (Klenow enzyme) of pol I family, complexed with substrates, has been resolved, b) significant information regarding the process of substrate and template-primer binding by pol I has been obtained from kinetic analysis, c) some of the sites (amino acid residues) participating in the substrates and template binding have been identified, d) a number of catalytic residues with some functional implication have been identified by site-directed mutagenesis, and e) this enzyme serves as the model system for mechanistic study of all DNA polymerases. In order to identify and relate important structural domains that carry out specific function in the catalysis of DNA synthesis, the following tripartite approach will be used: i) site directed mutagenesis of amino acid residues in conserved domains or implied by 3-D model structure examinations. An in depth analysis of the properties of mutant enzyme will clarify the role for the desired amino acid in specific domain structure, ii) photo-affinity labeling of enzyme proteins with template-primers and identification of sites of enzyme and template-primer contact and, iii) utilize all available structural information concerning DNA polymerases in the interpretation of mutagenesis results as well as to construct structural models which provide the detailed functional participation of various domain structures in atomic details and permit structural elucidation of the transition state, of the catalytic reaction. The molecular mechanism and functional anatomy of DNA polymerase clarified in this manner will lead to a better understanding of DNA replication, DNA repair and mutagenic effects of chemicals and carcinogenesis.

描述（改编自申请人的摘要）：DNA的酶促合成 是一个多步过程，需要底物的顺序结合， 伴随着酶蛋白中的几种构象变化。这 这些步骤中涉及的分子机制尚不清楚。这 该类酶的许多晶体结构的最新可用性， 但是，基本分子取得了重大进步 DNA聚合的机制及其与结构的关系 可以在原子水平上阐明酶的构成。主要目标 该建议是继续研究生化，酶学 原型酶的结构特性，即大肠杆菌聚合酶1。 在拟议的研究中选择该酶是基于以下事实。 pol I的大片段（klenow酶）的三维解剖结构 已经解决了与底物复合的家庭，b）显着 有关底物和模板 - 提示器绑定过程的信息 从动力学分析获得了pol i，c）一些位点（氨基酸 残留物）参与基板和模板结合已 识别，d）许多具有功能含义的催化残基 已通过位置定向诱变来识别，e）该酶是 所有DNA聚合酶的机械研究模型系统。为了 识别并关联进行特定的重要结构域 在DNA合成的催化中的功能，以下三方方法 将使用：i）位于保守的氨基酸残基的定向诱变 域或由3-D模型结构检查暗示。深入分析 突变酶的特性将阐明所需氨基的作用 特定域结构中的酸，ii）酶的光亲和力标记 蛋白质具有模板提示物，并鉴定酶的位点和 模板 - 播种机触点和iii）利用所有可用的结构信息 关于诱变结果的解释中的DNA聚合酶 为了构建提供详细功能的结构模型 各种领域结构参与原子细节并许可 过渡状态的结构阐明，催化反应。这 在此阐明的DNA聚合酶的分子机制和功能解剖结构 方式将使对DNA复制，DNA修复和 化学物质和癌变的诱变作用。