DYNAMICS OF DNA RECOGNITION BY DNA REPAIR POLYMERASE

DNA 修复聚合酶识别 DNA 的动力学

• 批准号: 6351267
• 负责人: W. M. BUJALOWSKI
• 金额: $ 21.4万
• 依托单位: UNIVERSITY OF TEXAS MED BR GALVESTON
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-02-01 至 2003-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6351267
• 关键词: DNA; DNA directed DNA polymerase; DNA repair; analytical ultracentrifugation; chemical kinetics; conformation; enzyme structure; enzyme substrate complex; fluorescence resonance energy transfer; intermolecular interaction; molecular dynamics; nucleic acid structure; nucleoside triphosphate; physical model; site directed mutagenesis; solutions; thermodynamics

DNA replication and repair are fundamental processes for transmission of genetic information from one cell generation to the other and for defending the cell against damages in its DNA. At the heart of these processes is the synthesis of DNA catalyzed by the DNA polymerases. The mammalian polymerase beta provides an outstanding model system to study the molecular mechanism of the polymerase action due to its simplified catalytic repertoire and its role in mammalian DNA repair. Pol beta is one of the four recognized DNA-directed polymerases of the eucaryotic nucleus. The enzyme is involved in gap filling synthesis, in mismatch repair, repair of monofunctional adducts, UV damaged DNA, and abasic lesions in DNA. In light of its key role in mammalian DNA repair, it is of fundamental importance to understand the molecular mechanism by which pol beta functions. Knowledge of the mechanistic details of the polymerase mechanism is essential to our understanding of the DNA repair process in a human cell and the mechanism by which the cell defends itself against diseases, e.g., cancer. Studying different steps on the molecular level should provide the necessary knowledge about how to control these processes. In turn, this knowledge will be very useful in designing efficient therapies for diseases. DNA repair polymerase is designed to perform DNA synthesis on gapped DNA with vanishing gap size which suggests that the mode of enzyme interactions with nucleic acids is changing, in the course of DNA synthesis. Polymerase beta lacks error correcting activities, typical for replicative polymerases, which indicates that DNA and dNTP recognition processes, which control fidelity of DNA synthesis, precede the chemical step. Thus, elucidation of the energetics and dynamics of the substrate recognition process by pol beta, including the transitions between different binding modes, is a prerequisite for understanding the molecular mechanism of the enzyme action, particularly, the fidelity of the DNA synthesis. The main goal of this project is to establish a molecular model of the recognition process of DNA and dNTP substrates by pol beta. This goal will be achieved through quantitative thermodynamic, kinetic, and structural studies of its complexes with DNA and dNTPs in solution using quantitative fluorescence titrations, analytical centrifugation methods, fluorescence stopped-flow, temperature-jump, rapid-quench-flow, fluorescence energy transfer, and site-directed mutagenesis techniques.

DNA复制和修复是传播的基本过程 遗传信息从一代细胞到另一代细胞， 保护细胞免受DNA损伤。在这些问题的核心 DNA聚合酶催化的DNA合成过程。 哺乳动物聚合酶β提供了一个出色的模型系统， 研究聚合酶作用的分子机制， 简化的催化库及其在哺乳动物DNA修复中的作用。 Pol β是四种公认的DNA定向聚合酶之一， 真核细胞 该酶参与间隙填充合成， 在错配修复、单功能加合物修复、UV损伤DNA 以及DNA中的碱基损伤。 鉴于其在哺乳动物DNA修复中的关键作用，它是至关重要的 重要的是要了解分子机制， 功能协调发展的 聚合酶的机械细节知识 这一机制对于我们理解DNA修复过程至关重要 以及细胞自我保护的机制 对抗疾病，例如，癌 学习不同的步骤 分子水平应该提供必要的知识， 控制这些过程。 反过来，这些知识将非常有用 设计有效的疾病疗法。 DNA修复聚合酶被设计用于在有缺口的DNA上进行DNA合成 间隙大小消失，这表明酶的模式 与核酸的相互作用是不断变化的， 合成. 聚合酶β缺乏纠错活性，典型的 对于复制型聚合酶，这表明DNA和dNTP 识别过程，控制DNA合成的保真度， 化学步骤。 因此，阐明的能量学和动力学的 通过pol β的底物识别过程，包括跃迁 在不同的绑定模式之间，是理解 酶作用的分子机制，特别是， DNA合成 该项目的主要目标是建立一个分子模型， pol β对DNA和dNTP底物的识别过程。这一目标 将通过定量的热力学，动力学， 在溶液中与DNA和dNTPs复合物的结构研究 定量荧光滴定，分析离心方法， 荧光停止流动、温度跳跃、快速猝灭流动、 荧光能量转移和定点诱变技术。