MECHANISM AND FIDELITY OF DNA REPLICATION

DNA复制发生的速度很快，并且高度 忠诚度，这两者都是 有机体。这项提议的长期目标是结构性的， DNA高保真高效的动力学和热力学基础 首席调查员的初步数据表明 快速化学猝灭流动法测定T7DNA聚合酶途径。在……里面 此外，pI已经构建并完全缺乏核酸外切酶 T7 DNA聚合酶突变体，使定量检测成为可能 个体反应对数据保真度的贡献 聚合反应。这些研究为这一提议奠定了坚实的基础。 具体目标是：(1)探索 通过考察动力学和热力学来实现选择性 作为DNA序列变异的函数的掺入，通过 使用核苷酸类似物，并通过检查位置- 模板/底物中不匹配的情况。(2)叙述的结构 酶的完全定量对催化动力学的影响 单一和多个氨基酸的动力学和热力学结果 替换；最初，定点突变将由 与T7 DNA高度同源的DNA聚合酶I的结构 聚合酶，但最终我们希望我们的研究建立在晶体的基础上 T7DNA聚合酶的结构。(3)开展研究，建立 T7 DNA聚合酶和基因4蛋白解旋酶/启动酶的结构 X射线结晶学，以开始解决问题 聚合反应的催化机理和分子保真度 这可能与反应动力学有关。(4)建立 基因4蛋白解旋酶和底物酶反应的动力学， 包括三磷酸腺苷水解酶与DNA偶联途径的定义 RNA引物基本步骤的解开和检验 综合。(5)通过以下方法表征复制叉处的DNA聚合 DNA聚合酶化学计量学的建立及应用 复制复合体中的解旋酶/起始酶，通过检查 快速化学急冷法合成超前滞后链的动力学 流动方法，并通过快速光化学交联研究 建立复合体中蛋白质-蛋白质相互作用的动力学。 这些研究将提供基本的机理信息，描述 DNA复制保真度，对理解 真核聚合酶和艾滋病毒逆转录酶。

DNA replication occurs at fast rates and with a high degree of fidelity, both of which are necessary for the survival and propagation of organisms. The long term objective of this proposal is the structural, kinetic, and thermodynamic basis for high fidelity and efficiency of DNA Preliminary data by the principal investigator has established the pathway of T7 DNA polymerase by rapid chemical-quench-flow methods. In addition, the PI has constructed and fully char exonuclease deficient mutant of T7 DNA polymerase which has enabled quantitative measurement o the contributions of individual reactions to the fidelity of polymerization. These studies lay a strong foundation for this proposal. The specific aims are to: (1) Explore the structural basis for selectivity by examination of the kinetics and thermodynamics of incorporation as a function of variations in DNA sequence, through the use of nucleotide analogs, and by examination of the effect of the posi- tion of mismatches in the template/primer. (2) Relate the structure of the enzyme to the dynamics of catalysis by complete quantitation of the kinetic and thermodynamic consequences of single and multiple amino acid substitutions; initially, site-directed mutagenesis will be guided by the structure of DNA polymerase I, which is highly homologous to T7 DNA polymerase, but ultimately we hope to base our studies on the crystal structures of T7 DNA polymerase. (3) Begin studies to establish the structure of T7 DNA polymerase and gene 4 protein helicase/primase by x-ray crystallography in order to begin to address the problems of catalytic mechanism and fidelity of polymerization in molecular terms that can be related to the kinetics of reaction. (4) Establish the kinetics of the helicase and primase reactions of gene 4 protein, including definition of the pathway of coupling of ATP hydrolysis to DNA unwinding and examination of the elementary steps leading to RNA primer synthesis. (5) Characterize DNA polymerization at a replication fork by establishing the stoichiometries of DNA polymerase and the helicase/primase in the replication complex, by examination of the kinetics of leading and lagging strand synthesis by rapid chemical-quench flow methods, and by rapid photochemical crosslinking studies to establish the dynamics of protein-protein associations in the complex. These studies will provide fundamental mechanistic information describing DNA replication fidelity, with important implications for understanding eukaryotic polymerases and HIV reverse transcriptase.