Cargando…
Microhomology-mediated end joining is the principal mediator of double-strand break repair during mitochondrial DNA lesions
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 (nDN...
Autores principales: | , , , , , |
---|---|
Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
The American Society for Cell Biology
2016
|
Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4713127/ https://www.ncbi.nlm.nih.gov/pubmed/26609070 http://dx.doi.org/10.1091/mbc.E15-05-0260 |
_version_ | 1782410148849909760 |
---|---|
author | Tadi, Satish Kumar Sebastian, Robin Dahal, Sumedha Babu, Ravi K. Choudhary, Bibha Raghavan, Sathees C. |
author_facet | Tadi, Satish Kumar Sebastian, Robin Dahal, Sumedha Babu, Ravi K. Choudhary, Bibha Raghavan, Sathees C. |
author_sort | Tadi, Satish Kumar |
collection | PubMed |
description | 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. |
format | Online Article Text |
id | pubmed-4713127 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2016 |
publisher | The American Society for Cell Biology |
record_format | MEDLINE/PubMed |
spelling | pubmed-47131272016-03-30 Microhomology-mediated end joining is the principal mediator of double-strand break repair during mitochondrial DNA lesions Tadi, Satish Kumar Sebastian, Robin Dahal, Sumedha Babu, Ravi K. Choudhary, Bibha Raghavan, Sathees C. Mol Biol Cell Articles 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. The American Society for Cell Biology 2016-01-15 /pmc/articles/PMC4713127/ /pubmed/26609070 http://dx.doi.org/10.1091/mbc.E15-05-0260 Text en © 2016 Tadi et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0). “ASCB®,” “The American Society for Cell Biology®,” and “Molecular Biology of the Cell®” are registered trademarks of The American Society for Cell Biology. |
spellingShingle | Articles Tadi, Satish Kumar Sebastian, Robin Dahal, Sumedha Babu, Ravi K. Choudhary, Bibha Raghavan, Sathees C. Microhomology-mediated end joining is the principal mediator of double-strand break repair during mitochondrial DNA lesions |
title | Microhomology-mediated end joining is the principal mediator of double-strand break repair during mitochondrial DNA lesions |
title_full | Microhomology-mediated end joining is the principal mediator of double-strand break repair during mitochondrial DNA lesions |
title_fullStr | Microhomology-mediated end joining is the principal mediator of double-strand break repair during mitochondrial DNA lesions |
title_full_unstemmed | Microhomology-mediated end joining is the principal mediator of double-strand break repair during mitochondrial DNA lesions |
title_short | Microhomology-mediated end joining is the principal mediator of double-strand break repair during mitochondrial DNA lesions |
title_sort | microhomology-mediated end joining is the principal mediator of double-strand break repair during mitochondrial dna lesions |
topic | Articles |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4713127/ https://www.ncbi.nlm.nih.gov/pubmed/26609070 http://dx.doi.org/10.1091/mbc.E15-05-0260 |
work_keys_str_mv | AT tadisatishkumar microhomologymediatedendjoiningistheprincipalmediatorofdoublestrandbreakrepairduringmitochondrialdnalesions AT sebastianrobin microhomologymediatedendjoiningistheprincipalmediatorofdoublestrandbreakrepairduringmitochondrialdnalesions AT dahalsumedha microhomologymediatedendjoiningistheprincipalmediatorofdoublestrandbreakrepairduringmitochondrialdnalesions AT baburavik microhomologymediatedendjoiningistheprincipalmediatorofdoublestrandbreakrepairduringmitochondrialdnalesions AT choudharybibha microhomologymediatedendjoiningistheprincipalmediatorofdoublestrandbreakrepairduringmitochondrialdnalesions AT raghavansatheesc microhomologymediatedendjoiningistheprincipalmediatorofdoublestrandbreakrepairduringmitochondrialdnalesions |