MITOCHONDRIAL DNA DAMAGE AND REPAIR

线粒体DNA(MtDNA)突变会导致许多人类疾病，如MELAS(线粒体脑病、乳酸酸中毒和中风)和LHON(Leber遗传性视神经病变)，并可能导致其他更常见的疾病，包括帕金森氏症和糖尿病。这些突变反映了线粒体DNA修复过程的不足，并表明需要更详细地了解线粒体的DNA修复过程。最近的研究表明，某些类型的损伤，主要是那些易于碱基切除修复的损伤，可以在mtDNA中修复。碱基切除修复途径处理由自发碱基丢失或由几种损伤特异性糖基酶中的任何一种产生的mtDNA中的基本位置。一些参与DNA氧化损伤修复的糖基酶的核基因为细胞核和线粒体提供了酶。我们实验室最近用从线粒体中提纯的蛋白质重建了线粒体DNA碱基切除修复的完整途径。我们建议对作用于3-甲基腺嘌呤残基的线粒体糖基化酶进行实验，以验证线粒体含有细胞型烷基腺嘌呤糖基化酶的假设。这种酶还可以处理脂质过氧化产物与线粒体DNA反应所产生的内源性损伤。碱性位点修复需要一种酶来切割碱性位点，即AP(无嘌呤/脱嘧啶)核酸内切酶。我们还提出了一系列实验来表征线粒体AP内切酶。最后，我们建议应用我们在mtDNA修复方面的专业知识来研究一种已知导致帕金森病的环境毒素干扰mtDNA复制的启动的机制。我们将检验这一假设，即这种抑制是由于DNA聚合酶伽马在DNA损伤部位的隔离造成的。

Mutations in mitochondrial DNA (mtDNA) cause a number of human diseases such as MELAS (mitochondrial encephalopathy lactic acidosis and stroke) and LHON (Leber's hereditary optic neuropathy) and may contribute to the pathogenesis of other more common diseases, including Parkinson's disease and diabetes. These mutations reflect an inadequacy of mtDNA repair processes and indicate that more detailed understanding of DNA repair processes in mitochondria is required. Recent studies have shown that some types of damage, mainly those amenable to base excision repair, can be repaired in mtDNA. The base excision repair pathway handles abasic sites in mtDNA produced by spontaneous base loss or by any of several damage-specific glycosylases. Some nuclear genes for glycosylases involved in repair of oxidative DNA damage provide enzyme to both the nucleus and to mitochondria. Our laboratory has recently reconstituted a complete pathway for base excision repair of mtDNA with proteins purified from mitochondria. We propose experiments to characterize mitochondrial glycosylases that act on 3- methyl adenine residues in order to test the hypothesis that mitochondrial contain a variant of the cellular alkyl adenine glycosylase. This enzyme may also process endogenous damage generated by reaction of lipid peroxidation products with mtDNA. Abasic site repair requires an enzyme to cleave abasic sites, AP (apurinic/apyrimidinic) endonuclease. We also propose a series of experiments to characterize the mitochondrial AP endonuclease. Finally, we propose to apply our expertise in mtDNA repair to investigate the mechanism whereby an environmental toxin known to cause Parkinson's disease interferes with the initiation of mtDNA replication. We will test the hypothesis that this inhibition results from the sequestration of DNA polymerase gamma at sites of DNA damage.