Elementary Steps in DNA Polymerization

描述（由申请人提供）：本提案的长期目标是定义高保真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。控制核苷酸选择性的反应中的基本步骤将通过使用瞬态动力学方法（停流和化学猝灭流）结合定点诱变和位点特异性标记的全面和严格的动力学分析来定义。T7 DNA聚合酶将被用作模型系统，因为它代表了最好的可用高保真聚合酶，具有E-DNA-核苷酸三元复合物的高分辨率晶体结构，准备进行催化。将建立核苷酸诱导的蛋白质结构变化的动力学，以及活性位点残基的这种重要重排在DNA复制的特异性和效率中的作用。将检查DNA聚合酶活性位点和校正核酸外切酶活性位点之间的动力学分配，包括分析蛋白质中结构元件的作用，这些结构元件在聚合酶位点处检测错配，以及稳定单链DNA在核酸外切酶位点处的结合和催化。这项工作将提供DNA聚合酶在复制DNA中实现这种非凡保真度的机制的全面图片。这项工作很重要，因为它将为负责核苷酸识别和识别错配或核苷酸类似物的关键事件提供标准。研究结果将被应用于提高我们对一些癌症、衰老和与线粒体相关的遗传性疾病起源的理解！DNA复制错误，病毒聚合酶对核苷类似物的选择性掺入，以及用于治疗病毒感染的核苷类似物的毒副作用。这项工作提供的基本分子细节将为理解系统中的核苷酸选择性提供基础，这些系统可能不适合这种详细的分析，并且与这些重要的人类健康问题有因果关系。

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.