Krebs Institute and Sheffield Institute for Nucleic Acids, Department of Molecular Biology and Biotechnology, Firth Court, University of Sheffield, S10 2TN Sheffield, U.K.
Wellcome Trust Centre for Mitochondrial Research, Medical School, Newcastle University, Framlington Place, Newcastle upon Tyne NE2 4HH, U.K.
Sci Adv. 2017 Apr 28;3(4):e1602506. doi: 10.1126/sciadv.1602506. eCollection 2017 Apr.
Breakage of one strand of DNA is the most common form of DNA damage. Most damaged DNA termini require end-processing in preparation for ligation. The importance of this step is highlighted by the association of defects in the 3'-end processing enzyme tyrosyl DNA phosphodiesterase 1 (TDP1) and neurodegeneration and by the cytotoxic induction of protein-linked DNA breaks (PDBs) and oxidized nucleic acid intermediates during chemotherapy and radiotherapy. Although much is known about the repair of PDBs in the nucleus, little is known about this process in the mitochondria. We reveal that TDP1 resolves mitochondrial PDBs (mtPDBs), thereby promoting mitochondrial gene transcription. Overexpression of a toxic form of mitochondrial topoisomerase I (TOP1mt*), which generates excessive mtPDBs, results in a TDP1-dependent compensatory up-regulation of mitochondrial gene transcription. In the absence of TDP1, the imbalance in transcription of mitochondrial- and nuclear-encoded electron transport chain (ETC) subunits results in misassembly of ETC complex III. Bioenergetics profiling further reveals that TDP1 promotes oxidative phosphorylation under both basal and high energy demands. It is known that mitochondrial dysfunction results in free radical leakage and nuclear DNA damage; however, the detection of intermediates of radical damage to DNA is yet to be shown. Consequently, we report an increased accumulation of carbon-centered radicals in cells lacking TDP1, using electron spin resonance spectroscopy. Overexpression of the antioxidant enzyme superoxide dismutase 1 (SOD1) reduces carbon-centered adducts and protects TDP1-deficient cells from oxidative stress. Conversely, overexpression of the amyotrophic lateral sclerosis-associated mutant SOD1 leads to marked sensitivity. Whereas Tdp1 knockout mice develop normally, overexpression of SOD1 suggests early embryonic lethality. Together, our data show that TDP1 resolves mtPDBs, thereby regulating mitochondrial gene transcription and oxygen consumption by oxidative phosphorylation, thus conferring cellular protection against reactive oxygen species-induced damage.
DNA 单链断裂是最常见的 DNA 损伤形式。大多数受损的 DNA 末端需要进行末端处理，为连接做准备。3'-端加工酶酪氨酸 DNA 磷酸二酯酶 1（TDP1）缺陷与神经退行性变的相关性，以及化疗和放疗过程中蛋白连接的 DNA 断裂（PDBs）和氧化核酸中间产物的细胞毒性诱导，凸显了这一步骤的重要性。尽管人们对核内 PDBs 的修复了解很多，但对线粒体中这一过程的了解却很少。我们揭示 TDP1 可解决线粒体 PDBs（mtPDBs），从而促进线粒体基因转录。表达一种有毒形式的线粒体拓扑异构酶 I（TOP1mt*），它会产生过多的 mtPDBs，导致 TDP1 依赖性线粒体基因转录的代偿性上调。在没有 TDP1 的情况下，线粒体和核编码电子传递链（ETC）亚基的转录失衡会导致 ETC 复合物 III 组装错误。生物能谱分析进一步表明，TDP1 在基础和高能量需求下均能促进氧化磷酸化。众所周知，线粒体功能障碍会导致自由基泄漏和核 DNA 损伤；然而，自由基对 DNA 损伤的中间产物的检测尚未得到证实。因此，我们使用电子自旋共振光谱法报告了 TDP1 缺失细胞中碳中心自由基的积累增加。抗氧化酶超氧化物歧化酶 1（SOD1）的过表达减少了碳中心加合物，并保护 TDP1 缺陷细胞免受氧化应激。相反，与肌萎缩侧索硬化症相关的突变 SOD1 的过表达导致明显的敏感性。虽然 Tdp1 敲除小鼠发育正常，但 SOD1 的过表达表明早期胚胎致死性。总之，我们的数据表明 TDP1 解决 mtPDBs，从而调节线粒体基因转录和氧消耗通过氧化磷酸化，从而赋予细胞对活性氧诱导的损伤的保护作用。
