MECHANISM AND FIDELITY OF DNA REPLICATION

这项提案的目标是建立结构性、动力性和 DNA复制的保真度和效率的热力学基础。 对艾滋病毒逆转录酶的分析将允许定义 最终可能限制病毒能力的结构性限制 避免核苷类似物在治疗中的适当组合 艾滋病和其他病毒感染。线粒体DNA的分析 聚合酶将确定核苷类似物毒性的来源 被认为是由于类似物被合并到线粒体DNA中。 对T7DNA聚合酶和HIV RT的研究表明， 聚合酶是两步核苷酸结合反应的函数 涉及由核苷酸引起的构象从“开放”到 三元E.DNA.dNTP复合体的“克隆”状态。以水晶为基础 HIV RT与DNA的复合结构，已经建立了一个工作模型 提出了运动的“手指”领域的建议 聚合酶进入DNA主沟对a结合的反应 正确的碱基对。这项建议的具体目的是：(1) 确定与构象有关的蛋白质结构域 变化导致紧密的核苷酸结合和快速聚合； 将使用特定的光亲和标记来定位氨基酸 它们与下面的DNA主沟紧密接触 构象变化。(2)现场的暂态动力学分析 将使用定向突变体来进一步定义在 “封闭”构象状态和量化的贡献 单独的氨基酸。构象变化的动力学将是 通过使用荧光标记的核苷酸的停流法检测， DNA和蛋白质。(3)DNA结构对构象的影响 将考察聚合反应的变化和动力学。在……里面 特别是，DNA弯曲的作用将被研究，其机制 移码突变的研究将通过测量 延伸到突变前移码中间体之上。(4)的影响 RT催化聚合反应动力学中的RNA二级结构 将会被研究，并且核衣壳蛋白对 将评估聚合酶阅读发夹的能力。(5) 线粒体聚合酶的基因将被克隆，条件是 优化活性蛋白的高效表达和纯化。 线粒体聚合酶的机制和保真度将是 通过动力学测量来确定。

The goal of this proposal is to establish the structural, kinetic and thermodynamic basis for the fidelity and efficiency of DNA replication. Analysis of HIV reverse transcriptase will allow definition of the structural constraints that ultimately may limit the ability of the virus to avoid an appropriate combination of nucleoside analogs in the treatment of AIDS and other viral infections. Analysis of the mitochondrial DNA polymerase will define the origins of the toxicity of nucleoside analogs thought to be due to incorporation of the analogs into mitochondrial DNA. Studies on T7 DNA polymerase and HIV RT have shown that the selectivity of the polymerase is a function of a two-step nucleotide binding reaction involving a nucleotide-induced change in conformation from an "open" to a "cloned" state of the ternary E.DNA.dNTP complex. Based upon the crystal structure of HIV RT complexed with DNA, a working model has been formulated proposing the movement of the "fingers" domain of the polymerase into the major groove of DNA in response to the binding of a correct base pair. The specific aims of this proposal are to: (1.) Identify the protein structural domains involved in the conformational change leading to tight nucleotide binding and rapid polymerization; specific photo-affinity labeling will be used to localize amino acids which are brought into close contact with the DNA major groove following the conformational change. (2.) Transient state kinetic analysis of site- directed mutants will be employed to further define the contacts made in the "closed" conformational state and to quantify the contributions of individual amino acids. The kinetics of the conformational change will be examined by stopped-flow methods using fluorescently labeled nucleotides, DNA and protein. (3.) The effects of DNA structure on the conformational change and the kinetics of polymerization will be examined. In particular, the role of DNA bends will be investigated, and the mechanisms of frameshift mutagenesis will be examined by measuring the kinetics of extension over premutational frameshift intermediates. (4.) The effect of RNA secondary structure on the kinetics of polymerization catalyzed by RT will be investigated and the effect of the nucleocapsid protein on the ability of the polymerase to read through hairpins will be assessed. (5.) Genes for the mitochondrial polymerase will be cloned and conditions will be optimized for the overexpression and purification of active protein. The mechanism and fidelity of the mitochondrial polymerase will be established by kinetic measurements.