Dynamics of DNA Recognition by DNA Repair Polymerase

DNA 修复聚合酶识别 DNA 的动力学

• 批准号: 7012332
• 负责人: W. M. BUJALOWSKI
• 金额: $ 26.45万
• 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 2007-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7012332
• 关键词: DNA directed DNA polymerase; DNA repair; analytical ultracentrifugation; binding sites; chemical kinetics; chemical models; enzyme mechanism; enzyme structure; enzyme substrate complex; fluorescence resonance energy transfer; fluorescent dye /probe; nucleic acid structure; site directed mutagenesis; thermodynamics

DESCRIPTION (provided by applicant): 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. Polymerase 13 provides an outstanding model system to study the molecular mechanism of the polymerase action, due to its simplified catalytic repertoire and role in human DNA repair. The enzyme is involved in gap filling synthesis, mismatch repair, the repair of monofunctional adducts, UV damaged DNA, and abasic lesions in DNA. Mutations and deletions in pol 13 have been implicated in several human cancers and genetic diseases including breast, prostate, kidney, lung, colorectal cancers, and Wemer's syndrome. In light of its key role in human DNA repair, it is of fundamental importance to understand the molecular mechanism by which pol 13 functions in performing its activities. Knowledge of the mechanistic details of the enzyme 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. Studying different steps at the molecular level will provide the knowledge about how to control them. In turn, this knowledge will be of paramount importance in designing efficient therapies for diseases. Polymerase 13 lacks error-correcting activities typical for replicative polymerases. This indicates that DNA and dNTP recognition, which controls the fidelity of DNA syntheses, must precede the catalysis. Moreover, a profound difference between the replicative and repair polymerase is that the DNA repair enzyme must recognize a specific structure of the damaged DNA prior to the catalysis, in the context of overwhelmingly dsDNA conformation. Thus, elucidations of the energetics and dynamics of pol 13-DNA recognition processes and the structures of the formed complexes are prerequisites for understanding the molecular mechanism of the enzyme, particularly, the efficiency and fidelity of the DNA synthesis. The main goal of this project is to establish a molecular model of the recognition of specific DNA structures by pol 13. This goal will be achieved through quantitative thermodynamic, kinetic, and structural studies of its complexes with DNA substrates and dNTPs in solution, using fluorescence titrations, analytical centrifugation, fluorescence stopped-flow, temperature-jump, rapid-quench-flow, fluorescence energy transfer, and site-directed mutagenesis techniques.

描述（由申请人提供）：DNA复制和修复是遗传信息从一代传递到另一代的基本过程，也是保护细胞免受DNA损伤的基本过程。这些过程的核心是DNA聚合酶催化的DNA合成。由于聚合酶13在人类DNA修复中的简化催化功能和作用，它为研究聚合酶作用的分子机制提供了一个杰出的模型系统。该酶参与间隙填充合成、错配修复、单功能加合物的修复、紫外线损伤的DNA和DNA中的基本损伤。pol 13的突变和缺失与几种人类癌症和遗传疾病有关，包括乳腺癌、前列腺癌、肾癌、肺癌、结直肠癌和韦默氏综合征。鉴于其在人类DNA修复中的关键作用，了解pol 13在执行其活动中的分子机制具有重要意义。了解酶机制的机制细节对我们理解人类细胞中的DNA修复过程和细胞防御疾病的机制至关重要。在分子水平上研究不同的步骤将提供如何控制它们的知识。反过来，这些知识对于设计有效的疾病治疗方法将是至关重要的。聚合酶13缺乏典型的复制聚合酶的纠错活性。这表明DNA和控制DNA合成保真度的dNTP识别必须先于催化。此外，复制聚合酶和修复聚合酶之间的一个深刻区别是，在绝大多数双链DNA构象的情况下，DNA修复酶必须在催化之前识别受损DNA的特定结构。因此，阐明pol - 13-DNA识别过程的能量学和动力学以及形成的复合物的结构是理解该酶的分子机制，特别是DNA合成的效率和保真度的先决条件。本项目的主要目标是建立pol - 13识别特定DNA结构的分子模型。这一目标将通过荧光滴定、分析离心、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，对其与DNA底物和dNTPs在溶液中的复合物进行定量热力学、动力学和结构研究来实现。