Mechanism and Fidelity of DNA Replication

1992年，FDA批准了ddC （zalcitabine）用于治疗艾滋病，这是在新的加速审查程序下获得批准的第一种药物，但现在已知ddC具有严重的毒副作用，因此今天很少使用。在1993年的一项乙型肝炎临床试验中，5名患者死于另一种类似物FIAU的毒副作用。基于我们在过去拨款期间的发现，我们现在可以在进入临床之前识别出那些最有可能产生线粒体毒性的化合物。核苷类似物的毒性与其线粒体DNA聚合酶的掺入率和校对外切酶的去除率有关。直接速率测量表明，目前批准用于治疗艾滋病的七种核苷/核苷酸类似物的毒性差异超过10万倍。尽管其他作用位点可能导致一些观察到的毒副作用，但这项工作提供了一个易于量化的系统来评估线粒体毒性的潜力，并提供了一种检测方法来筛选HIV RT或其他病毒聚合酶的新抑制剂的潜在毒性。本研究的目的是建立控制核苷酸特异性的结构和机制规则，以便我们可以预测哪些类似物可能毒性最小。特别是，新的特征，可能会刺激去除校对外切酶可能导致更有效和毒性更小的药物。核苷酸选择性的结构和机制基础将通过对线粒体聚合酶的快速反应动力学研究、突变分析和结构/功能研究的结合来检验。快速反应动力学分析将允许对天然和非天然核苷酸的掺入进行定量分析，从而根据经验绘制掺入和去除反应期间施加在核苷酸上的结构约束。突变酶的分析和酶的结构/功能研究将有助于探索核苷酸选择性的酶结构基础。此外，我们将研究线粒体DNA聚合酶在碱基切除修复中的作用及其对氧化损伤DNA的反应。预计这项工作将为设计和测试具有较低人类毒性的新型核苷类似物提供基础，用于治疗病毒感染，并提供控制线粒体DNA复制的反应的基本知识。

DESCRIPTION: In 1992 the FDA approved ddC (zalcitabine) for the treatment of AIDS, the first drug to receive approval under the new accelerated review process but it is now known that ddC has severe toxic side effects and is rarely used today. In a 1993 hepatitis B clinical trial, five patients died from toxic side effects of another analog, FIAU. Based upon the discoveries we made during the past grant period we can now identify those compounds with the highest potential for mitochondrial toxicity before they go into the clinic. The toxicity of nucleoside analogs is correlated with their rate of incorporation by the mitochondrial DNA polymerase and their rate of removal by the proofreading exonuclease. Direct rate measurements indicate greater than a 100,000-fold variation in toxicity among the seven nucleoside/nucleotide analogs currently approved for the treatment of AIDS. Although other sites of action may lead to some observed toxic side effects, this work provides an easily quantifiable system to assess the potential for mitochondrial toxicity and provides an assay to screen for potential toxicity of new inhibitors of HIV RT or other viral polymerases. The goal of this research is to establish the structural and mechanistic rules governing nucleotide specificity for incorporation and removal so that we can predict which analogs are likely to be the least toxic. In particular, new features that may stimulate removal by the proofreading exonuclease could lead to even more effective and less toxic drugs. The structural and mechanistic basis for nucleotide selectivity will be examined using a combination of rapid reaction kinetic studies, mutation analysis, and structure/function studies on the mitochondrial polymerase. The rapid reaction kinetic analysis will allow quantification of the incorporation of natural and unnatural nucleotides to map empirically the structural constrains imposed upon the nucleotides during incorporation and excision reactions. Analysis of mutant enzymes and structure/function studies on the enzyme will afford exploration of the enzymatic structural basis for nucleotide selectivity. In addition, we will examine the roles of the mitochondrial DNA polymerase in base excision repair and its response to oxidatively damaged DNA. It is anticipated that this work will provide the basis for the design and testing of new nucleoside analogs with lower human toxicity to be used in the treatment of viral infections as well as providing basic knowledge of the reactions governing the replication of mitochondrial DNA.