The Mechanism of Mitochondrial DNA Proofreading in Human Health and Disease

摘要 线粒体有自己的闭合环状基因组。线粒体DNA(MtDNA)的突变率是核DNA的10倍。由于DNA聚合酶伽马是线粒体内唯一已知的DNA聚合酶，它对于线粒体DNA的忠实复制、校对和修复是必不可少的。这项建议的总体目标是了解线粒体DNA代谢中DNA聚合酶γ外切酶活性的基本机制，并确定其在疾病中的作用。基于以下假设，我们假设DNA聚合酶γ的核外溶解活性改变会导致线粒体功能障碍和疾病。首先，DNA聚合酶伽马核酸外切酶区域的突变会导致毁灭性的线粒体疾病。 其次，DNA聚合酶伽马的聚合酶结构域突变导致容易出错的表型，可以通过功能性核酸外切酶来改善。第三，校对缺陷DNA聚合酶伽马的小鼠模型显示线粒体DNA突变增强，衰老表型加速，寿命缩短。第四，用于控制HIV感染的核苷逆转录酶抑制剂(NRTI)通过抑制DNA聚合酶伽马而导致线粒体功能障碍。 线粒体疾病可由遗传和环境因素引起。不幸的是，没有治愈或有效的长期治疗方法。随着越来越多的疾病表现出线粒体缺陷，我们必须了解缺陷是如何导致疾病的。我们将通过以下方式研究DNA聚合酶γ的核酸外切酶结构域在维持线粒体DNA完整性中的作用： 目的1.研究核酸外切酶区域内疾病突变的潜在后果，以及这些突变是如何导致线粒体功能障碍和疾病的。 目的2.确定核酸外切酶结构域中参与校对的关键区域，并确定核酸外切酶剔除NRTI的效率，以及在模板中错误地结合相反的错误编码损伤(如氧化应激的标志物8oxoG)和环境DNA损伤(如苯并[a]芘引起的损伤)的效率。

ABSTRACT Mitochondria have their own closed circular genome. Mitochondrial DNA (mtDNA) has a 10 times greater rate of mutation than nuclear DNA. As DNA polymerase gamma is the only known DNA polymerase within the mitochondrion, it is essential for faithful replication, proofreading and repair of mtDNA. The overall goal of this proposal is to understand the fundamental mechanisms of exonuclease activity of DNA polymerase gamma in mtDNA metabolism, and to define its role in disease. Based on the following, we hypothesize that altered exonucleolytic activity of DNA polymerase gamma leads to mitochondrial dysfunction and disease. First, mutations in the exonuclease domain of DNA polymerase gamma cause devastating mitochondrial diseases. Second, polymerase domain mutations in DNA polymerase gamma that cause an error-prone phenotype can be ameliorated by functional exonuclease. Third, mouse models of proofreading-deficient DNA polymerase gamma show enhanced mtDNA mutations and accelerated ageing phenotype with reduced lifespan. Fourth, nucleoside reverse transcriptase inhibitors (NRTIs) used to control HIV infection induce mitochondrial dysfunction by inhibiting DNA polymerase gamma. Mitochondrial diseases can be caused by genetic and environmental factors. Unfortunately, there is no cure or no effective long-term treatment. As more diseases show mitochondrial defects, it is imperative that we understand how defects lead to disease. We will investigate the role of the exonuclease domain of DNA polymerase gamma in maintaining the integrity of mtDNA by: AIM 1. To investigate the underiying consequences of disease mutations within the exonuclease domain, and how these give rise to mitochondrial dysfunction and disease. AIM 2. To identify the critical regions within the exonuclease domain that are involved in proofreading, and to determine how efficientiy the exonuclease excises incorporated NRTIs, as well as nucleotides incorrectly incorporated opposite miscoding lesions in the template (such as 8oxoG, a marker of oxidative stress), and environmental DNA lesions (such as those caused by benzo[a]pyrene).