Mechanism and Fidelity of DNA Replication

描述：1992年，FDA批准了ddC(扎西他滨)用于治疗艾滋病，这是根据新的加速审查程序获得批准的第一种药物，但现在已知ddC有严重的毒副作用，今天很少使用。在1993年的一项乙肝临床试验中，5名患者死于另一种类似物FIAU的毒副作用。根据我们在过去的授权期内所做的发现，我们现在可以在进入临床之前识别那些具有最高线粒体毒性潜力的化合物。核苷类似物的毒性与它们被线粒体DNA聚合酶掺入的速率和它们被校对核酸外切酶去除的速率有关。直接比率测量表明，目前批准用于治疗艾滋病的七种核苷/核苷酸类似物之间的毒性差异超过100,000倍。虽然其他作用部位可能会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可能性，并提供了一种筛选HIV RT或其他病毒聚合酶新抑制剂的潜在毒性的方法。这项研究的目标是建立控制核苷酸特异性的结构和机制规则，以便我们能够预测哪些类似物可能是毒性最小的。特别是，可能会刺激校对核酸外切酶去除的新功能可能会导致更有效和毒性更低的药物。我们将结合快速反应动力学研究、突变分析和线粒体聚合酶的结构/功能研究来研究核苷酸选择性的结构和机制基础。快速反应动力学分析将允许量化天然和非天然核苷酸的掺入，以经验地绘制在掺入和切除反应期间施加在核苷酸上的结构约束。对突变酶的分析和对该酶的结构/功能的研究将为探索核苷酸选择性的酶结构基础提供依据。此外，我们还将研究线粒体DNA聚合酶在碱基切除修复中的作用及其对DNA氧化损伤的反应。预计这项工作将为设计和测试新的核苷类似物提供基础，这些核苷类似物具有较低的人类毒性，用于治疗病毒感染，并提供控制线粒体DNA复制反应的基本知识。

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.