Regulation of mitochondrial genome replication by hypoxia: The role of DNA oxidation in D-loop region.
Regulation of mitochondrial genome replication by hypoxia: The role of DNA oxidation in D-loop region.
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DOI：
10.1016/j.freeradbiomed.2016.04.011
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发表时间：
2016-07
影响因子：
7.4
通讯作者：
Ruchko MV
中科院分区：
文献类型：
--
作者：
Pastukh VM;Gorodnya OM;Gillespie MN;Ruchko MV
哺乳动物细胞的线粒体含有多个拷贝的线粒体DNA。虽然线粒体DNA拷贝数可以根据生理和病理生理条件而变化很大，但调控线粒体基因组复制的机制仍然不清楚。与促进生长、细胞增殖和线粒体生物生成的许多其他生理刺激一样，低氧也使用活性氧作为信号分子。新的证据表明，低氧诱导的核基因转录需要在启动子区域的特定序列中控制DNA损伤和修复。类似的机制是否在线粒体中起作用尚不清楚。在这里，我们检验了线粒体基因组D-环区受控的氧化DNA损伤和修复是线粒体DNA复制和转录所必需的假设。我们发现低氧对肺动脉内皮细胞线粒体蛋白的表达影响不大，但增加了线粒体DNA的含量。线粒体DNA拷贝数的增加伴随着线粒体基因组D-环区的氧化修饰。为了研究线粒体基因组的这种序列特异性氧化在mtDNA复制中的作用，我们在大鼠肺动脉内皮细胞中过表达线粒体靶向的8-氧鸟嘌呤糖基酶Ogg1，增强了转基因细胞的mtDNA修复能力。过表达Ogg1可抑制缺氧诱导的D-loop区线粒体DNA氧化，抑制缺氧诱导的线粒体DNA复制。Ogg1的过表达还减少了线粒体转录因子A(TFAM)与线粒体基因组调节区和编码区的结合，而不改变对照或低氧细胞中TFAM的总丰度。这些观察表明，缺氧期间D-loop区域的DNA氧化修饰对于增加TFAM结合和随后的线粒体基因组复制是重要的。
Mitochondria of mammalian cells contain multiple copies of mitochondrial (mt) DNA. Although mtDNA copy number can fluctuate dramatically depending on physiological and pathophysiologic conditions, the mechanisms regulating mitochondrial genome replication remain obscure. Hypoxia, like many other physiologic stimuli that promote growth, cell proliferation and mitochondrial biogenesis, uses reactive oxygen species as signaling molecules. Emerging evidence suggests that hypoxia-induced transcription of nuclear genes requires controlled DNA damage and repair in specific sequences in the promoter regions. Whether similar mechanisms are operative in mitochondria is unknown. Here we test the hypothesis that controlled oxidative DNA damage and repair in the D-loop region of the mitochondrial genome are required for mitochondrial DNA replication and transcription in hypoxia. We found that hypoxia had little impact on expression of mitochondrial proteins in pulmonary artery endothelial cells, but elevated mtDNA content. The increase in mtDNA copy number was accompanied by oxidative modifications in the D-loop region of the mitochondrial genome. To investigate the role of this sequence-specific oxidation of mitochondrial genome in mtDNA replication, we overexpressed mitochondria-targeted 8-oxoguanine glycosylase Ogg1 in rat pulmonary artery endothelial cells, enhancing the mtDNA repair capacity of transfected cells. Overexpression of Ogg1 resulted in suppression of hypoxia-induced mtDNA oxidation in the D-loop region and attenuation of hypoxia-induced mtDNA replication. Ogg1 overexpression also reduced binding of mitochondrial transcription factor A (TFAM) to both regulatory and coding regions of the mitochondrial genome without altering total abundance of TFAM in either control or hypoxic cells. These observations suggest that oxidative DNA modifications in the D-loop region during hypoxia are important for increased TFAM binding and ensuing replication of the mitochondrial genome.