Suter M, Richter C
Laboratory of Biochemistry I, Swiss Federal Institute of Technology, Zürich, Switzerland.
Biochemistry. 1999 Jan 5;38(1):459-64. doi: 10.1021/bi9811922.
Previous analyses indicated a high level of oxidative base modification in mitochondrial DNA, the extent of which raised questions about the methodological validity and biological implications. In the present study DNA was isolated from rat liver mitochondria under carefully controlled conditions, and the extent of base oxidation, DNA fragmentation, and nuclear DNA contamination were analyzed. DNA isolated from intact mitochondria treated with DNase consisted of 16.3 kilobase pairs, mostly circular, mitochondrial DNA molecules and a mixture of nuclear and mitochondrial DNA fragments, as identified by agarose gel electrophoresis and hybridization. High-performance liquid chromatography in combination with electrochemical detection confirmed that the overall level of 8-hydroxy-2'-deoxyguanosine, a marker commonly used in the analysis of base oxidation, is higher in mitochondrial than in nuclear DNA. Importantly, 8-hydroxy-2'-deoxyguanosine is relatively scarce in the 16.3 kilobase pair mitochondrial DNA molecules (0.051 pmol/microgram) but is present in high levels in mitochondrial DNA fragments (0.741 pmol/microgram). The fragments constitute about 18% of total mitochondrial DNA. The antitumor agent bleomycin, which binds to DNA, forms an iron complex capable of transferring electrons from Fe2+ to molecular oxygen. Exposure of mitochondria to bleomycin and iron resulted in nicking but not in a significant increase in base oxidation of 16.3 kilobase pair mitochondrial DNA, whereas the amount and the oxidation level of fragmented mitochondrial DNA significantly increased. These findings are relevant for a better understanding of the role of mitochondria in aging and various diseases and are consistent with the notion that despite the overall high DNA oxidation level, mitochondria can faithfully proliferate.
先前的分析表明线粒体DNA中存在高水平的氧化性碱基修饰，其程度引发了关于方法学有效性和生物学意义的问题。在本研究中，在严格控制的条件下从大鼠肝脏线粒体中分离出DNA，并分析了碱基氧化程度、DNA片段化和核DNA污染情况。经琼脂糖凝胶电泳和杂交鉴定，用DNA酶处理完整线粒体后分离出的DNA由16.3千碱基对组成，主要是环状线粒体DNA分子以及核DNA和线粒体DNA片段的混合物。高效液相色谱结合电化学检测证实，碱基氧化分析中常用的标志物8-羟基-2'-脱氧鸟苷在线粒体中的总体水平高于核DNA。重要的是，8-羟基-2'-脱氧鸟苷在16.3千碱基对的线粒体DNA分子中相对较少（0.051皮摩尔/微克），但在线粒体DNA片段中含量很高（0.741皮摩尔/微克）。这些片段约占线粒体DNA总量的18%。与DNA结合的抗肿瘤药物博来霉素形成一种能够将电子从Fe2+转移到分子氧的铁复合物。线粒体暴露于博来霉素和铁会导致16.3千碱基对线粒体DNA产生切口，但碱基氧化没有显著增加，而片段化线粒体DNA的量和氧化水平则显著增加。这些发现有助于更好地理解线粒体在衰老和各种疾病中的作用，并且与尽管DNA氧化总体水平较高但线粒体仍能忠实增殖的观点一致。
