Laboratory of Molecular Gerontology, National Institute on Aging, NIH, Baltimore, MD 21236, USA.
Methods. 2010 Aug;51(4):416-25. doi: 10.1016/j.ymeth.2010.02.020. Epub 2010 Feb 25.
The main source of mitochondrial DNA (mtDNA) damage is reactive oxygen species (ROS) generated during normal cellular metabolism. The main mtDNA lesions generated by ROS are base modifications, such as the ubiquitous 8-oxoguanine (8-oxoG) lesion; however, base loss and strand breaks may also occur. Many human diseases are associated with mtDNA mutations and thus maintaining mtDNA integrity is critical. All of these lesions are repaired primarily by the base excision repair (BER) pathway. It is now known that mammalian mitochondria have BER, which, similarly to nuclear BER, is catalyzed by DNA glycosylases, AP endonuclease, DNA polymerase (POLgamma in mitochondria) and DNA ligase. This article outlines procedures for measuring oxidative damage formation and BER in mitochondria, including isolation of mitochondria from tissues and cells, protocols for measuring BER enzyme activities, gene-specific repair assays, chromatographic techniques as well as current optimizations for detecting 8-oxoG lesions in cells by immunofluorescence. Throughout the assay descriptions we will include methodological considerations that may help optimize the assays in terms of resolution and repeatability.
线粒体 DNA（mtDNA）损伤的主要来源是正常细胞代谢过程中产生的活性氧（ROS）。ROS 产生的主要 mtDNA 损伤是碱基修饰，如普遍存在的 8-氧鸟嘌呤（8-oxoG）损伤；然而，碱基缺失和链断裂也可能发生。许多人类疾病都与 mtDNA 突变有关，因此维持 mtDNA 完整性至关重要。所有这些损伤主要通过碱基切除修复（BER）途径修复。现在已知哺乳动物线粒体具有 BER，它与核 BER 类似，由 DNA 糖苷酶、AP 内切酶、DNA 聚合酶（线粒体中的 POLgamma）和 DNA 连接酶催化。本文概述了测量线粒体中氧化损伤形成和 BER 的程序，包括从组织和细胞中分离线粒体、测量 BER 酶活性的方案、基因特异性修复测定、色谱技术以及通过免疫荧光检测细胞中 8-oxoG 损伤的当前优化。在整个测定描述中，我们将包括方法学考虑因素，这些因素可能有助于根据分辨率和可重复性优化测定。
