STRUCTURE AND FUNCTION OF E COLI POLA GENE

本研究的总体目标是确定分子机制 DNA聚合酶通过它进行精确的和可加工的模板- 定向DNA合成。 E. coli DNA聚合酶I， 作为唯一的DNA聚合酶，高分辨率的结构数据， 提供了一个很好的模型系统， 由其他更复杂的聚合酶催化的反应。 克列诺碎片 两种酶活性，聚合酶和3 '-5'核酸外切酶， 起到校对的作用。 这两个活跃的网站位于 分离分子的结构域。 在我们对克列诺的研究中 片段，我们建议使用生物化学，结构和 基因方法，继续我们与 晶体群T.史泰茨 对于3 '-5'核酸外切酶，我们建议（i）测试反应机理 从我们以前的研究中提出，特别是假设， 攻击亲核试剂是金属缔合的氢氧根离子;（ii）改善 具有催化活性的酶-底物复合物的结构模型; 以及（iii）确定DNA结合位点的位置和程度 与核酸外切作用有关。 对于聚合酶活性位点，我们 应使用定点诱变和突变体的生化分析 蛋白质以扩展底物中重要残基的鉴定 结合、催化和转运。 为了解决机制问题， 负责聚合酶保真度，我们将筛选突变体与 突变表型和研究的生化特性， 突变酶 应用我们对聚合酶催化碱基错误掺入的了解 反应，我们将尝试开发一种局部诱变程序， 这将产生一个广泛的单一氨基酸的变化， 频率. 该程序将用于针对特定区域的 Klenow片段，补充定点诱变策略。 将仔细分析Klenow片段与DNA的相互作用。 足迹和磷酸盐乙基化干扰将用于 确定与双链DNA的复合物的尺寸和几何形状。 我们 将研究引物末端下游接触的作用， 确定Klenow片段和完整片段的底物偏好 DNA聚合酶I。 我们将研究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。 使用“高- 决议”足迹，我们将试图区分前， 以及引物末端在聚合酶处的易位后位置 活性部位 为了促进晶体学研究，特别是克列诺的研究， 与双链DNA片段复合物，我们将适当地构建 Klenow片段的工程衍生物。

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.