Elementary Steps in DNA Polymerization

描述(申请人提供)：本提案的长期目标是确定高保真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的基本步骤将通过使用暂态动力学方法(停流和化学猝灭流)、结合定点突变和定点标记进行全面和严格的动力学分析来确定。T7DNA聚合酶将被用作模型系统，因为它代表了可用的最好的高保真聚合酶，具有E-DNA-核苷酸三元复合体的高分辨率晶体结构，准备进行催化。核苷酸诱导蛋白质结构变化的动力学，以及这种重要的活性部位残基重排在DNA复制的特异性和效率中的作用，将被确立。将研究DNA聚合酶活性位点和校对核酸外切酶活性位点之间的动力学分配，包括分析蛋白质中检测到聚合酶位点错配的结构元素的作用，以及那些稳定核酸外切酶位点单链DNA结合和催化的结构元素的作用。这项工作将提供DNA聚合酶在复制DNA时实现如此非凡的保真度的机制的全面图景。这项工作很重要，因为它将为负责核苷酸识别和错配或核苷酸类似物区分的关键事件提供标准。这些结果将应用于改善我们对某些癌症、衰老和与线粒体有关的遗传性疾病的起源过程的理解！DNA复制错误，病毒聚合酶选择性地掺入核苷类似物，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，这些系统可能无法进行如此详细的分析，并与这些重大的人类健康问题存在因果关系。

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.