STRUCTURE AND FUNCTION OF E COLI POLA GENE

本研究的总体目标是确定分子机制。 DNA聚合酶通过它来执行准确和连续的模板- 直接合成DNA。大肠杆菌DNA聚合酶I的Klenow片段， 作为唯一的DNA聚合酶，其高分辨率结构数据 提供了一个极好的模型系统来理解 由其他更复杂的聚合酶催化的反应。Klenow碎片有 两种酶活性，聚合酶和3‘-5’核酸外切酶 起到校对的作用。这两个活跃的站点位于 分离分子的结构域。在我们对克利诺的研究中 片段，我们建议使用生化、结构和 基因方法，继续我们与 T.Steitz的结晶学群。 对于3‘-5’核酸外切酶，我们建议(I)检验反应机理 从我们以前的研究中提出的，特别是假设 攻击亲核试剂是一种金属缔合的氢氧离子；(Ii)改善 催化活性酶-底物复合体的结构模型； 以及(Iii)确定DNA结合部位的位置和范围 与核外溶解作用有关。对于聚合酶活性位点，我们 应使用定点突变和突变体的生化分析 蛋白质扩大底物中重要残基的鉴定 结合、催化和易位。为了解决这些机制 负责聚合酶的保真度，我们将用一种 诱变子表型及其生化特性的研究 突变的酶。 应用我们对聚合酶催化的碱基错掺入的知识 反应，我们将尝试开发一种局部诱变程序 这应该会在很高的时间内产生一种单一氨基酸的广泛变化 频率该程序将用于针对 Klenow片段，补充了定点突变策略。 Klenow片段与DNA的相互作用将被仔细分析。 足迹和磷酸乙基化干扰将用于 用双链DNA确定复合体的尺寸和几何形状。我们 应调查引子末端下游的触点在 Klenow片段和完整的底物偏好的测定 DNA聚合酶I.我们将检查DNA之间的重叠程度 聚合酶和核酸外切酶反应中使用的结合模式。用“高-- 解决方案“足迹我们将尝试区分前 以及聚合酶上的引物末端的易位后位置 活动站点。 为了促进结晶学研究，特别是Klenow的研究 与双链DNA的片段复合体，我们应该适当地构建 克雷诺碎片的工程衍生品。

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.