Structure and Function of DNA Polymerase I of E.coli

大肠杆菌DNA聚合酶I的结构和功能

• 批准号: 6890870
• 负责人: NIGEL David GRINDLEY
• 金额: $ 54.36万
• 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 2007-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6890870
• 关键词: Archaea; DNA directed DNA polymerase; DNA repair; DNA replication; Escherichia coli; X ray crystallography; active sites; bacterial DNA; bacterial genetics; bacterial proteins; conformation; enzyme activity; enzyme mechanism; enzyme structure; fluorescence; gene mutation; hydrogen bond; intermolecular interaction; nucleic acid sequence; phenotype; protein structure function; species difference

DESCRIPTION (provided by investigator): The overall goal of this project is a full understanding, at the molecular level, of the reactions catalyzed by DNA polymerases, with particular emphasis on how polymerases ensure substrate specificity and accuracy in copying DNA. The question of polymerase accuracy has important health implications because the errors made by DNA polymerases can result in mutations leading to human disease. Moreover, DNA polymerases are frequently targeted in chemotherapeutic and antiviral strategies, as well as being important in a variety of diagnostic biotechnology applications, so an understanding of their reaction mechanisms is crucial. Experiments are proposed on two model DNA polymerases that have contrasting enzymatic properties: the highly accurate DNA polymerase I (Klenow fragment) o E. coli, and the much less accurate Dbh bypass polymerase from the archaeon S. solfataricus. Structural data are available for both these enzymes and several close homologues, and serve as the basis for many of the planned experiments. Moreover, because the important features of the polymerase active site and reaction mechanism are conserved throughout the polymerase family, the results obtained with these simple model systems will have much wider relevance. A major priority will be the investigation of noncovalent steps in the polymerase reaction pathway. These include the early steps involved in substrate recognition and rearrangement of the active site into a form poised for chemical catalysis, and the translocation step that must occur to vacate the active site for the next cycle of addition. A variety of fluorescence assays will be used, in combination with rapid single-turnover kinetics, with the goals of identifying the physical processes involved and understanding their roles in the specificity of the reaction. Fidelity mechanisms at the polymerase active site will be explored using mutants of Klenow fragment. Nucleotide analogues will be used to investigate the role of base pair shape and hydrogen-bonding interactions in the specificity of both Klenow fragment and Dbh polymerase. Mechanistic probes developed for Klenow fragment will be applied to studies of Dbh, in order to learn more about the reaction mechanism and the role of active site side chains in this recently discovered error-prone polymerase. This enzyme has a remarkable propensity for frameshifting, which may provide clues as to its in vivo function, perhaps indicating an ability to bypass certain types of bulky DNA lesions.

描述（研究人员提供）：该项目的总体目标是在分子水平上完全理解DNA聚合酶催化的反应，特别强调了聚合酶如何确保复制DNA的底物特异性和准确性。聚合酶准确性的问题具有重要的健康影响，因为DNA聚合酶的误差会导致突变导致人类疾病。此外，DNA聚合酶经常针对化学治疗和抗病毒策略，并且在各种诊断生物技术应用中很重要，因此对它们的反应机制的理解至关重要。对具有对比酶促特性的两个模型DNA聚合酶提出了实验：高度精确的DNA聚合酶I（klenow片段）O大肠杆菌，以及来自Archaeon S. solfataricus的精确的DBH旁路聚合酶。这些酶和几种紧密的同源物都可以使用结构数据，并作为许多计划实验的基础。此外，由于聚合酶活性位点和反应机制的重要特征在整个聚合酶家族中保守，因此这些简单模型系统获得的结果将具有更大的相关性。主要优先级是研究聚合酶反应途径中非共价步骤。其中包括涉及底物识别和重新排列的早期步骤，使其成为有准备化学催化的形式，以及必须发生的易位步骤，以使活跃位点撤离下一个添加周期。将使用多种荧光测定，结合快速的单转动力学，以及识别所涉及的物理过程并了解其在反应特异性中的作用的目标。聚合酶活性位点的富达机制将使用Klenow片段的突变体进行探索。核苷酸类似物将用于研究碱基对形状和氢键相互作用在Klenow片段和DBH聚合酶的特异性中的作用。为Klenow片段开发的机械探针将应用于DBH的研究，以便更多地了解反应机理和活性位点链链在最近发现的易用错误的聚合酶中的作用。该酶对壁画具有显着的倾向，这可能提供了有关其体内功能的线索，也许表明能够绕过某些类型的笨重的DNA病变。