Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Warsaw, Poland.
Mutat Res. 2012 Dec;740(1-2):21-33. doi: 10.1016/j.mrfmmm.2012.12.004. Epub 2012 Dec 28.
Oxidative stress is known to enhance the frequency of two major types of alterations in the mitochondrial genome of Saccharomyces cerevisiae: point mutations and large deletions resulting in the generation of respiration-deficient petite rhō mutants. We investigated the effect of antimycin A, a well-known agent inducing oxidative stress, on the stability of mtDNA. We show that antimycin enhances exclusively the generation of respiration-deficient petite mutants and this is accompanied by a significant increase in the level of reactive oxygen species (ROS) and in a marked drop of cellular ATP. Whole mitochondrial genome sequencing revealed that mtDNAs of antimycin-induced petite mutants are deleted for most of the wild-type sequence and usually contain one of the active origins of mtDNA replication: ori1, ori2 ori3 or ori5. We show that the frequency of antimycin-induced rhō mutants is significantly elevated in mutants deleted either for the RAD50 or XRS2 gene, both encoding the components of the MRX complex, which is known to be involved in the repair of double strand breaks (DSBs) in DNA. Furthermore, enhanced frequency of rhō mutants in cultures of antimycin-treated cells lacking Rad50 was further increased by the simultaneous absence of the Ogg1 glycosylase, an important enzyme functioning in mtBER. We demonstrate also that rad50Δ and xrs2Δ deletion mutants display a considerable reduction in the frequency of allelic mitochondrial recombination, suggesting that it is the deficiency in homologous recombination which is responsible for enhanced rearrangements of mtDNA in antimycin-treated cells of these mutants. Finally, we show that the generation of large-scale mtDNA deletions induced by antimycin is markedly decreased in a nuc1Δ mutant lacking the activity of the Nuc1 nuclease, an ortholog of the mammalian mitochondrial nucleases EndoG and ExoG. This result indicates that the nuclease plays an important role in processing of oxidative stress-induced lesions in the mitochondrial genome.
点突变和大片段缺失，导致呼吸缺陷型 petite rhō 突变体的产生。我们研究了抗霉素 A，一种已知诱导氧化应激的试剂，对 mtDNA 稳定性的影响。我们表明，抗霉素 A 仅增强呼吸缺陷型 petite 突变体的产生，这伴随着活性氧 (ROS) 水平的显著增加和细胞 ATP 的明显下降。全线粒体基因组测序显示，抗霉素诱导的 petite 突变体的 mtDNA 缺失了大部分野生型序列，通常包含一个活跃的 mtDNA 复制起点：ori1、ori2、ori3 或 ori5。我们表明，RAD50 或 XRS2 基因缺失突变体中抗霉素诱导的 rhō 突变体的频率显著升高，RAD50 和 XRS2 基因分别编码 MRX 复合物的组成部分，该复合物已知参与 DNA 双链断裂 (DSB) 的修复。此外，在缺乏 Rad50 的抗霉素处理细胞的培养物中，增强 rhō 突变体的频率通过同时缺乏 Ogg1 糖苷酶进一步增加，Ogg1 糖苷酶是一种在 mtBER 中起重要作用的酶。我们还证明，rad50Δ 和 xrs2Δ 缺失突变体显示出等位基因线粒体重组频率的显著降低，表明正是同源重组的缺陷导致这些突变体的抗霉素处理细胞中线粒体 DNA 的重排增加。最后，我们表明，缺乏 Nuc1 核酸酶活性的 nuc1Δ 突变体中，由抗霉素诱导的大片段 mtDNA 缺失的产生明显减少，Nuc1 核酸酶是哺乳动物线粒体核酸酶 EndoG 和 ExoG 的同源物。这一结果表明，该核酸酶在处理氧化应激诱导的线粒体基因组损伤中起着重要作用。
