Functional Dynamics of Mammalian and Viral DNA Repair Polymerases

哺乳动物和病毒 DNA 修复聚合酶的功能动力学

• 批准号: 8029567
• 负责人: W. M. BUJALOWSKI
• 金额: $ 29.64万
• 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 2013-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8029567
• 关键词: ASFV pol X; Address; African Swine Fever Virus; Analytical Centrifugation; Binding; Binding Sites; Biological Models; Breast; Catalysis; Cells; Chemicals; Colorectal Cancer; Complex; DNA; DNA Binding; DNA Damage; DNA Repair; DNA Repair Enzymes; DNA Structure; DNA biosynthesis; DNA-Directed DNA Polymerase; Data; Deletion Mutation; Disease; Effectiveness; Energy Transfer; Enzymes; Fluorescence; Fluorescence Anisotropy; Functional disorder; Generations; Genetic; Goals; Heart; Hereditary Disease; Human; Human Genetics; Infection; Kidney; Kinetics; Knowledge; Lead; Light; Lung; Malignant Neoplasms; Malignant neoplasm of lung; Mammalian Cell; Methods; Modeling; Molecular; Molecular Conformation; Mutation; Nature; Nucleic Acids; Nucleotides; Polymerase; Process; Prostate; Reaction; Research; Research Project Grants; Resistance; Role; Series; Site-Directed Mutagenesis; Solutions; Staging; Structure; System; Techniques; Testing; Thermodynamics; Time; Titrations; Virus; Virus Diseases; Werner Syndrome; base; cancer genetics; conformational conversion; design; enzyme mechanism; fight against; gene therapy; human DNA; mutant; physical model; repaired; response; stopped-flow fluorescence; transmission process; viral DNA

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 or against viral infections. At the heart of these processes is the synthesis of the DNA catalyzed by DNA polymerases. Mammalian Polymerase (3 (pol p) and African Swine Fever Virus Polymerase X (pol X) provide outstanding model systems to study the molecular mechanism of the DNA repair polymerase action due to its simplified structures and catalytic repertoires. Because of the fundamental role in human DNA repair and the virus defense against the host reaction to the infection, pol P and pol X are enzymatic systems of a paramount biomedical importance. Mutations and deletions in pol p have been implicated in several human cancers and genetic diseases including breast, prostate, kidney, lung, colorectal cancers, and Werner syndrome. Mutations in pol X render the virus vulnerable to the DNA- modifying apparatus of the cell, which weakens the progress of the virus infection. In light of the pol (3 key role in human DNA repair and the pol X essential role in the effectiveness of viral infection of the mammalian cell, it is of fundamental importance to understand the molecular mechanism by which pol p and pol X function in performing their activities. Knowledge of mechanistic details of the mechanisms is essential to our understanding of the DNA repair processes in a human cell, the mechanism by which the cell defends itself against diseases, and the mechanism by which the cell fights against viral infections. Studying different steps at the molecular level will provide the necessary knowledge about how to control them. In turn, this knowledge is invaluable for designing rational and efficient therapies for genetic, cancer and viral diseases. The profound and fundamental difference between the replicative and repair polymerases 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. This indicates that DNA and dNTP recognition, which controls fidelity of DNA synthesis, must precede the catalysis. Thus, elucidation of the energetics, dynamics and structure of pol p - DNA and ASFV pol X - DNA complexes is a prerequisite for understanding the molecular mechanisms of the enzymes, particularly, the efficiency and fidelity of catalysis. The main goal of this project is to elucidate the molecular mechanisms of the recognition of specific DNA structures by pol p and pol X and their role in DNA synthesis. This goal will be achieved through quantitative thermodynamic, kinetic, and structural studies of their complexes with DNA substrates and dNTPs in solution using quantitative fluorescence titrations, analytical centrifugation, fluorescence stopped-flow, 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损伤或病毒感染的基本过程。这些过程的核心是DNA聚合酶催化的DNA合成。哺乳动物聚合酶3 （pol p）和非洲猪瘟病毒聚合酶X （pol X）因其结构简化和催化谱的特点，为研究DNA修复聚合酶的分子机制提供了很好的模型系统。由于pol - P和pol - X在人类DNA修复和病毒防御宿主对感染反应中的基本作用，它们是具有重要生物医学意义的酶系统。pol - p的突变和缺失与多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、结直肠癌和沃纳综合征。pol X的突变使病毒容易受到细胞DNA修饰装置的攻击，从而削弱了病毒感染的进展。鉴于pol(3)在人类DNA修复中的关键作用以及pol X在病毒感染哺乳动物细胞的有效性中所起的重要作用，了解pol p和pol X发挥其活性的分子机制具有重要意义。了解这些机制的机理细节对于我们理解人类细胞中的DNA修复过程、细胞防御疾病的机制以及细胞抵抗病毒感染的机制至关重要。在分子水平上研究不同的步骤将为如何控制它们提供必要的知识。反过来，这些知识对于设计合理有效的基因、癌症和病毒性疾病的治疗方法是无价的。复制聚合酶和修复聚合酶之间深刻而根本的区别在于，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。这表明DNA和控制DNA合成保真度的dNTP识别必须先于催化。因此，阐明pol - p - DNA和ASFV pol - X - DNA复合物的能量学、动力学和结构是理解酶的分子机制，特别是催化效率和保真度的先决条件。本项目的主要目的是阐明pol p和pol X识别特定DNA结构的分子机制及其在DNA合成中的作用。这一目标将通过定量荧光滴定、分析离心、荧光停止流、快速猝灭流、荧光能量转移和定点诱变技术，对它们与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。