Microhomology-mediated end joining is the principal mediator of double-strand break repair during mitochondrial DNA lesions.
Microhomology-mediated end joining is the principal mediator of double-strand break repair during mitochondrial DNA lesions.
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DOI：
10.1091/mbc.e15-05-0260
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发表时间：
2016-01-15
影响因子：
3.3
通讯作者：
Raghavan SC
中科院分区：
文献类型：
--
作者：
Tadi SK;Sebastian R;Dahal S;Babu RK;Choudhary B;Raghavan SC
哺乳动物线粒体双链断裂的修复依赖于微同源介导的末端连接（MMEJ）。经典的NHEJ在线粒体中检测不到。DNA连接酶III参与线粒体MMEJ，而不是连接酶IV或连接酶I。参与线粒体MMEJ的蛋白质机制包括CtIP、FEN1、连接酶III、MRE11和PARP1。线粒体DNA （mtDNA）缺失与各种线粒体疾病有关。在人类中发现的缺失是由短的、直接重复的线粒体DNA序列组成的；然而，这种DNA重排的机制尚未被阐明。与核DNA （nDNA）相比，mtDNA更容易受到氧化损伤，这可能导致双链断裂（dsb）。尽管对nDNA中的DSB修复进行了很好的研究，但线粒体中的修复机制尚未表征。在本研究中，我们通过体外和离体实验探讨了DSB在线粒体中的修复机制。而经典的NHEJ （C-NHEJ）是不可检测的，微同源介导的替代NHEJ有效地修复线粒体中的dsb。有趣的是，在具有5-，8-,10-,13-,16-，19-和22-nt微同源性的DNA底物中观察到强大的微同源介导的末端连接（MMEJ）。此外，MMEJ效率随同源序列长度的增加而提高。Western blotting、免疫沉淀和蛋白抑制实验表明CtIP、FEN1、MRE11和PARP1参与线粒体MMEJ。敲低研究与其他实验一起表明，DNA连接酶III，而不是连接酶IV或连接酶I，在线粒体MMEJ期间主要负责dsb的最终密封。这些观察结果强调了MMEJ在维持哺乳动物线粒体基因组完整性方面的核心作用，并且可能与许多人类线粒体疾病中观察到的缺失有关。
Repair of double-strand breaks in mammalian mitochondria depends on microhomology-mediated end joining (MMEJ). Classical NHEJ is not detectable in mitochondria. DNA ligase III, but not ligase IV or ligase I, is involved in mitochondrial MMEJ. The protein machinery involved in miitochondrial MMEJ includes CtIP, FEN1, ligase III, MRE11, and PARP1. Mitochondrial DNA (mtDNA) deletions are associated with various mitochondrial disorders. The deletions identified in humans are flanked by short, directly repeated mitochondrial DNA sequences; however, the mechanism of such DNA rearrangements has yet to be elucidated. In contrast to nuclear DNA (nDNA), mtDNA is more exposed to oxidative damage, which may result in double-strand breaks (DSBs). Although DSB repair in nDNA is well studied, repair mechanisms in mitochondria are not characterized. In the present study, we investigate the mechanisms of DSB repair in mitochondria using in vitro and ex vivo assays. Whereas classical NHEJ (C-NHEJ) is undetectable, microhomology-mediated alternative NHEJ efficiently repairs DSBs in mitochondria. Of interest, robust microhomology-mediated end joining (MMEJ) was observed with DNA substrates bearing 5-, 8-, 10-, 13-, 16-, 19-, and 22-nt microhomology. Furthermore, MMEJ efficiency was enhanced with an increase in the length of homology. Western blotting, immunoprecipitation, and protein inhibition assays suggest the involvement of CtIP, FEN1, MRE11, and PARP1 in mitochondrial MMEJ. Knockdown studies, in conjunction with other experiments, demonstrated that DNA ligase III, but not ligase IV or ligase I, is primarily responsible for the final sealing of DSBs during mitochondrial MMEJ. These observations highlight the central role of MMEJ in maintenance of mammalian mitochondrial genome integrity and is likely relevant for deletions observed in many human mitochondrial disorders.