STRUCTURE AND FUNCTION OF E COLI POLA GENE

大肠杆菌 POLA 基因的结构和功能

• 批准号: 2175207
• 负责人: NIGEL David GRINDLEY
• 金额: $ 33.16万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1980
• 资助国家: 美国
• 起止时间: 1980-05-01 至 1995-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2175207
• 关键词: DNA binding protein; DNA directed DNA polymerase; DNA footprinting; DNA replication; Escherichia coli; X ray crystallography; bacterial DNA; bacterial genetics; computer data analysis; enzyme mechanism; enzyme structure; enzyme substrate complex; gene expression; gene mutation; genetic mapping; mutant; nucleic acid sequence; phosphodiesterase I; protein engineering; protein sequence; protein structure function; radiotracer; restriction fragment length polymorphism; site directed mutagenesis; spleen exonuclease; structural genes

The overall goal of this research is to determine the molecular mechanism by which a DNA polymerase carries out accurate and processive template- directed DNA synthesis. The Klenow fragment of E. coli DNA polymerase I, as the only DNA polymerase for which high-resolution structural data are available, provides an excellent model system for understanding the reactions catalyzed by other more complex polymerases. Klenow fragment has two enzymatic activities, the polymerase and a 3'-5' exonuclease that serves a proofreading function. The two active sites are located on separate structural domains of the molecule. In our studies of Klenow fragment, we propose to use a combination of biochemical, structural and genetic approaches, continuing our close collaboration with the crystallographic group of T. Steitz. For the 3'-5' exonuclease, we propose (i) to test the reaction mechanism proposed from our previous studies, in particular the hypothesis that the attacking nucleophile is a metal-associated hydroxide ion; (ii) to improve the structural model of a catalytically competent enzyme-substrate complex; and (iii) to determine the location and extent of DNA binding sites relevant to exonucleolytic action. For the polymerase active site, we shall use site-directed mutagenesis and biochemical analysis of mutant proteins to extend the identification of residues important in substrate binding, catalysis and translocation. To address the mechanisms responsible for polymerase fidelity we shall screen for mutants with a mutator phenotype and study the biochemical properties of the resulting mutant enzymes. Applying our knowledge of polymerase-catalyzed base misincorporation reactions, we shall attempt to develop a localized mutagenesis procedure that should produce a wide spectrum of single amino acid changes at a high frequency. The procedure will be used to target specific regions of the Klenow fragment, complementing the site-directed mutagenesis strategy. The interaction of Klenow fragment with DNA will be carefully analyzed. Footprinting and phosphate ethylation interference will be used to determine the dimensions and geometry of the complex with duplex DNA. We shall investigate the role of contacts downstream of the primer terminus in determining the substrate preferences of both Klenow fragment and intact DNA polymerase I. We shall examine the extent of overlap between DNA binding modes used in polymerase and exonuclease reactions. Using "high- resolution" footprinting we shall attempt to distinguish between the pre- and post-translocation positions of a primer terminus at the polymerase active site. To facilitate the crystallographic studies, particularly those of Klenow fragment complexes with duplex DNA, we shall construct appropriately engineered derivatives of Klenow fragment.

这项研究的总体目标是确定分子机制 通过此DNA聚合酶可以执行准确和过程模板 - 定向DNA合成。 大肠杆菌DNA聚合酶I的Klenow片段， 作为高分辨率结构数据的唯一DNA聚合酶 可用，提供了一个出色的模型系统，用于了解 反应由其他更复杂的聚合酶催化。 klenow碎片具有 两种酶促活性，聚合酶和3'-5'外切酶， 提供校对功能。 两个活动站点位于 分子的单独结构结构域。 在我们对克洛诺的研究中 碎片，我们建议将生化，结构和 遗传方法，继续我们与 T. Steitz的晶体学组。 对于3'-5'外切酶，我们建议（i）测试反应机制 提出了我们以前的研究，特别是 攻击亲核试剂是一种金属相关的氢氧化离子。 （ii）改进 具有催化能力的酶 - 底物复合物的结构模型； （iii）确定DNA结合位点的位置和程度 与外核酸作用相关。 对于聚合酶活性位点，我们 应使用位置定向的诱变和突变体的生化分析 蛋白质扩展鉴定在底物中重要的残基的鉴定 结合，催化和易位。 解决机制 负责聚合酶保真度，我们将筛选具有A的突变体 突变器表型并研究所得的生化特性 突变酶。 运用我们对聚合酶催化的基础失物的了解 反应，我们将尝试制定局部诱变程序 这将产生广泛的单氨基酸变化 频率。 该过程将用于针对特定区域 klenow碎片，补充了位置定向的诱变策略。 Klenow片段与DNA的相互作用将仔细分析。 足迹和磷酸盐乙基化干扰将用于 确定用双链DNA的复合物的尺寸和几何形状。 我们 应研究引物末端下游的接触作用 确定Klenow片段和完整的底物偏好 DNA聚合酶I。我们将检查DNA之间的重叠程度 聚合酶和外丝酶反应中使用的结合模式。 使用“高 - 决议“足迹，我们将尝试区分 底漆末端在聚合酶的转换后位置 活性站点。 促进晶体学研究，尤其是克洛诺的研究 用双链DNA碎片复合物，我们将适当构建 克洛诺碎片的工程衍生物。