Elementary Steps in DNA Polymerization

DNA 聚合的基本步骤

• 批准号: 7031545
• 负责人: KENNETH ALLEN JOHNSON
• 金额: $ 27.61万
• 依托单位: UNIVERSITY OF TEXAS AT AUSTIN
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-04-01 至 2009-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7031545
• 关键词: DNA directed DNA polymerase; DNA replication; active sites; chemical kinetics; enzyme mechanism; enzyme structure; exonuclease; nucleotides; polymerization; stop flow technique

DESCRIPTION (provided by applicant): The long term goals of this proposal are to define the structural, kinetic, thermodynamic and mechanistic basis for nucleotide selectivity during DNA replication by a high fidelity DNA polymerase. Elementary steps in the reactions governing nucleotide selectivity will be defined by comprehensive and rigorous kinetic analysis using transient-state kinetic methods (stopped-flow and chemical-quench-flow), combined with site-directed mutagenesis and site-specific labeling. The T7 DNA polymerase will be used as a model system, because it represents the best available high fidelity polymerase with a high resolution crystal structure of the E-DNA-nucleotide ternary complex, poised to carry out catalysis. The kinetics of nucleotide-induced changes in protein structure, and the role of this important rearrangement of the active site residues in the specificity and efficiency of DNA replication, will be established. The kinetic partitioning between the DNA polymerase active site and the proofreading exonuclease active site will be examined, including analysis of the role of structural elements in the protein that sense mismatches at the polymerase site and those that stabilize the binding and catalysis of single-stranded DNA at the exonuclease site. This work will provide a comprehensive picture of the mechanisms by which DNA polymerases achieve such extraordinary fidelity in replicating DNA. The work is important, in that it will provide the standard for critical events responsible for nucleotide recognition and discrimination against mismatches or nucleotide analogs. The results will be applied to improve our understanding of processes underlying the origins of some cancers, aging and hereditary disorders related to mitochondria! DNA replication errors, the selective incorporation of nucleoside analogs by viral polymerases, and the toxic side effects of nucleoside analogs used to treat viral infections. The fundamental molecular details provided by this work will provide the basis for understanding nucleotide selectivity in systems that may not be amenable to such detailed analysis and are causally related to these significant human health issues.

描述（由申请人提供）：该提案的长期目标是定义通过高保真DNA聚合酶在DNA复制过程中核苷酸选择性的结构，动力学，热力学和机理基础。使用瞬态态动力学方法（停止流量和化学液分 - 流）结合位置定向的诱变和位点特异性标记，使用核苷酸选择性的反应中的基本步骤将通过全面和严格的动力学分析来定义。 T7 DNA聚合酶将用作模型系统，因为它代表了具有E-DNA-核苷酸三核三元复合物高分辨率晶体结构的最佳可用高富达聚合酶，该晶体有望进行催化。核苷酸诱导的蛋白质结构变化的动力学，将确定活性位点残基在DNA复制的特异性和效率中的重要重排的作用。将检查DNA聚合酶活性位点和校对外切酶活性位点之间的动力学分配，包括分析结构元素在蛋白质中的作用，这些元素在聚合酶位点感觉不匹配的蛋白质以及稳定外核酸酶单链DNA的结合和分解的蛋白质。这项工作将为DNA聚合酶在复制DNA中实现这种非凡的保真度提供全面的图景。这项工作很重要，因为它将为负责核苷酸识别和歧视不匹配或核苷酸类似物的关键事件提供标准。结果将用于提高我们对某些癌症，衰老和遗传性疾病起源的过程的理解，与线粒体有关！ DNA复制误差，通过病毒聚合酶选择性掺入核苷类似物以及用于治疗病毒感染的核苷类似物的毒性副作用。这项工作提供的基本分子细节将为理解可能不适合这种详细分析的系统中的核苷酸选择性提供基础，并且与这些重大的人类健康问题有因果关系。