title: Microhomology-mediated end joining is the principal mediator of double-strand break repair during mitochondrial DNA lesions.
abstract: 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.
authors.affiliation: Department of Biochemistry, Indian Institute of Science, Bangalore 560 012, India.
authors.forename: Satish Kumar
authors.initials: SK
authors.lastname: Tadi
authors.affiliation: Department of Biochemistry, Indian Institute of Science, Bangalore 560 012, India.
authors.forename: Robin
authors.initials: R
authors.lastname: Sebastian
authors.affiliation: Department of Biochemistry, Indian Institute of Science, Bangalore 560 012, India.
authors.forename: Sumedha
authors.initials: S
authors.lastname: Dahal
authors.affiliation: Department of Biochemistry, Indian Institute of Science, Bangalore 560 012, India.
authors.forename: Ravi K
authors.initials: RK
authors.lastname: Babu
authors.affiliation: Institute of Bioinformatics and Applied Biotechnology, Bangalore 560 100, India.
authors.forename: Bibha
authors.initials: B
authors.lastname: Choudhary
authors.affiliation: Department of Biochemistry, Indian Institute of Science, Bangalore 560 012, India sathees@biochem.iisc.ernet.in.
authors.forename: Sathees C
authors.initials: SC
authors.lastname: Raghavan
mesh_terms: D000818:Animals; D053903:DNA Breaks, Double-Stranded; D059766:DNA End-Joining Repair / Q000502:physiology*; D000072481:DNA Ligase ATP / Q000378:metabolism; D004272:DNA, Mitochondrial / Q000235:genetics* / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D008928:Mitochondria / Q000235:genetics / Q000378:metabolism; D051381:Rats; D059767:Recombinational DNA Repair
chemical_list: D004272:DNA, Mitochondrial; D004268:DNA-Binding Proteins; D000072481:DNA Ligase ATP