Mitochondrial DNA Oxidative Damage and Mutagenesis in Saccharomyces cerevisiae
Mitochondrial DNA Oxidative Damage and Mutagenesis in Saccharomyces cerevisiae
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DOI：
10.1007/978-1-59745-521-3_17
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发表时间：
2009-01-01
期刊：
影响因子：
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通讯作者：
Doetsch, Paul W.
中科院分区：
文献类型：
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作者：
Griffiths, Lyra M.;Doudican, Nicole A.;Doetsch, Paul W.
人类线粒体DNA(MtDNA)突变与母系遗传性神经肌肉疾病有关，并与更常见的疾病有关，如癌症、糖尿病和帕金森氏病。线粒体DNA的突变也会随着年龄的增长而积累，因此被认为是导致衰老和与年龄相关的病理的原因。线粒体DNA位于线粒体基质中，通过氧化磷酸化过程编码参与ATP生产的几种蛋白质，氧化磷酸化过程涉及高能电子通过电子传递链(ETC)流动。由于mtDNA靠近ETC，因此它很容易受到由该系统不断产生的过氧化氢、超氧化物和羟基自由基等活性氧物种(ROS)介导的氧化损伤。因此，能够在正常生理条件下以及在环境或疾病相关的应激状态下测量线粒体DNA氧化损伤是很重要的。酿酒酵母是一种单细胞真核兼性厌氧菌，其生存能力依赖于线粒体氧化磷酸化，因此是研究线粒体DNA氧化损伤的简便而又信息丰富的模型系统。在这里，我们描述了定量检测酿酒酵母线粒体DNA氧化损伤和突变的方法，其中一些方法可以应用于哺乳动物细胞和组织中类似的检测方法的开发。这些方法包括用红霉素耐药试验测量线粒体DNA中发生的点突变的数量，用改进的Southern印迹试验定量DNA氧化损伤的量，以及用“小诱导”试验测量线粒体DNA的完整性。
Mutation of human mitochondrial DNA (mtDNA) has been linked to maternally inherited neuromuscular disorders and is implicated in more common diseases Such as cancer, diabetes, and Parkinson's disease. Mutations in mtDNA also accumulate with age and arc therefore believed to contribute to aging and age-related pathology. Housed within the mitochondrial matrix, mtDNA encodes several of the proteins involved in the production of ATP via the process of oxidative phosphorylation, which involves the flow of high-energy electrons through the electron transport chain (ETC). Because Of its proximity to the ETC, mtDNA is highly vulnerable to oxidative damage mediated by reactive oxygen Species (ROS) Such as hydrogen peroxide, superoxide, and hydroxyl radicals that are constantly produced by this system. Therefore, it is important to be able to measure oxidative mtDNA damage under normal physiologic conditions and during environmental or disease-associated stress. The budding yeast, Saccharomyces cerevisiae, is a facile and informative model system in which to study Such mtDNA oxidative damage because it is a unicellular eukaryotic facultative anaerobe that is conditionally dependent on mitochondrial oxidative phosphorylation for viability. Here, we describe methods for quantifying oxidative mtDNA damage and mutagenesis in S. cerevisiae, several of which Could be applied to the development of similar assays in mammalian cells and tissues. These methods include measuring the number of point mutations that Occur in mtDNA with the erythromycin resistance assay, quantifying the amount of oxidative DNA damage utilizing a modified Southern blot assay, and measuring mtDNA integrity with the "petite induction" assay.