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: 7616682
• 负责人: NIGEL David GRINDLEY
• 金额: $ 58.95万
• 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 2011-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7616682
• 关键词: Accounting; Active Sites; Affect; Antiviral Agents; Archaea; Base Pairing; Binding; Biological Assay; Biological Models; Biotechnology; Bypass; Chemistry; Complex; DNA Adducts; DNA Modification Process; DNA Polymerase I; DNA Sequence; DNA-Directed DNA Polymerase; Data; Development; Diagnostic; Diphosphates; Disease; Ensure; Enzymes; Equilibrium; Escherichia coli; Event; Excision; Family; Fingers; Fluorescence; Goals; Grant; Health; Homologous Gene; Investigation; Kinetics; Lead; Learning; Lesion; Malignant Neoplasms; Measures; Metal Ion Binding; Modeling; Modern Medicine; Molecular; Motion; Movement; Mutation; Outcome; Pathway interactions; Pharmaceutical Preparations; Play; Polymerase; Process; Property; Reaction; Relative (related person); Reporter; Reporting; Research; Research Personnel; Role; Side; Specificity; Staging; Structure; Substrate Specificity; Techniques; Technology; Viral; Work; base; conformational conversion; design; experience; human disease; mutant; phosphodiester; programs; research study; tool

DESCRIPTION (provided by applicant): The overall goal of this project is a full understanding, at the molecular level, of the reactions catalyzed by DMA polymerases, with particular emphasis on how polymerases ensure substrate specificity and accuracy in copying DMA. The question of polymerase accuracy has important health implications because the errors made by DMA polymerases can result in mutations leading to human disease. Moreover, DMA 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. Our investigations focus on two model DMA polymerases that have contrasting enzymatic properties: the highly accurate DMA polymerase I (Klenow fragment) of 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 because these conformational transitions are likely to be involved in distinguishing between correctly paired substrates and the mispairs that result in polymerase errors. We will use fluorescence assays in combination with rapid single-turnover kinetics to investigate the rates of conformational changes and the effect on the reaction pathway of mispaired substrates, active site mutations and damaged DMA. DMA damage and errors in DMA synthesis can cause mutations that lead to diseases such as cancer, making it important to understand how DMA polymerases function to avoid these outcomes. Antiviral drugs frequently target viral polymerases so research into polymerase structures and mechanism is relevant in designing effective drugs and understanding how they work. DNA polymerases are also a crucial part of many of the diagnostic tools used in modern medicine and will be pivotal in the development of new DNA sequencing technologies for diagnostic purposes.

描述（由申请人提供）：该项目的总体目标是在分子水平上完全理解DMA聚合酶催化的反应，特别强调聚合酶如何确保复制DMA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DMA聚合酶的错误会导致突变导致人类疾病。此外，DMA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。我们的研究集中于两个具有对比酶促特性的模型DMA聚合酶：大肠杆菌的高度准确的DMA聚合酶I（Klenow片段），以及来自Archaeon S. solfataricus的精确型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。一个主要的优先级将是研究聚合酶反应途径中非共价步骤，因为这些构象转变很可能与区分正确配对的底物和导致聚合酶误差的错误相之间有关。我们将使用荧光测定与快速单转动力学结合使用荧光测定，以研究构象变化的速率以及对误导底物，主动位点突变和DMA损坏的反应途径的影响。 DMA损伤和DMA合成中的错误会导致突变导致癌症等疾病，从而了解DMA聚合酶如何发挥作用以避免这些结果。抗病毒药物经常靶向病毒聚合酶，因此对聚合酶结构和机制的研究与设计有效的药物和了解其工作方式相关。 DNA聚合酶也是现代医学中许多诊断工具的关键部分，并且在开发新的DNA测序技术方面将是诊断目的的关键部分。