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Microhomology-mediated end joining is activated in irradiated human cells due to phosphorylation-dependent formation of the XRCC1 repair complex

Microhomology-mediated end joining (MMEJ), an error-prone pathway for DNA double-strand break (DSB) repair, is implicated in genomic rearrangement and oncogenic transformation; however, its contribution to repair of radiation-induced DSBs has not been characterized. We used recircularization of a li...

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Autores principales: Dutta, Arijit, Eckelmann, Bradley, Adhikari, Sanjay, Ahmed, Kazi Mokim, Sengupta, Shiladitya, Pandey, Arvind, Hegde, Pavana M., Tsai, Miaw-Sheue, Tainer, John A., Weinfeld, Michael, Hegde, Muralidhar L., Mitra, Sankar
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Oxford University Press 2017
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5389627/
https://www.ncbi.nlm.nih.gov/pubmed/27994036
http://dx.doi.org/10.1093/nar/gkw1262
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author Dutta, Arijit
Eckelmann, Bradley
Adhikari, Sanjay
Ahmed, Kazi Mokim
Sengupta, Shiladitya
Pandey, Arvind
Hegde, Pavana M.
Tsai, Miaw-Sheue
Tainer, John A.
Weinfeld, Michael
Hegde, Muralidhar L.
Mitra, Sankar
author_facet Dutta, Arijit
Eckelmann, Bradley
Adhikari, Sanjay
Ahmed, Kazi Mokim
Sengupta, Shiladitya
Pandey, Arvind
Hegde, Pavana M.
Tsai, Miaw-Sheue
Tainer, John A.
Weinfeld, Michael
Hegde, Muralidhar L.
Mitra, Sankar
author_sort Dutta, Arijit
collection PubMed
description Microhomology-mediated end joining (MMEJ), an error-prone pathway for DNA double-strand break (DSB) repair, is implicated in genomic rearrangement and oncogenic transformation; however, its contribution to repair of radiation-induced DSBs has not been characterized. We used recircularization of a linearized plasmid with 3΄-P-blocked termini, mimicking those at X-ray-induced strand breaks, to recapitulate DSB repair via MMEJ or nonhomologous end-joining (NHEJ). Sequence analysis of the circularized plasmids allowed measurement of relative activity of MMEJ versus NHEJ. While we predictably observed NHEJ to be the predominant pathway for DSB repair in our assay, MMEJ was significantly enhanced in preirradiated cells, independent of their radiation-induced arrest in the G2/M phase. MMEJ activation was dependent on XRCC1 phosphorylation by casein kinase 2 (CK2), enhancing XRCC1's interaction with the end resection enzymes MRE11 and CtIP. Both endonuclease and exonuclease activities of MRE11 were required for MMEJ, as has been observed for homology-directed DSB repair (HDR). Furthermore, the XRCC1 co-immunoprecipitate complex (IP) displayed MMEJ activity in vitro, which was significantly elevated after irradiation. Our studies thus suggest that radiation-mediated enhancement of MMEJ in cells surviving radiation therapy may contribute to their radioresistance and could be therapeutically targeted.
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spelling pubmed-53896272017-04-24 Microhomology-mediated end joining is activated in irradiated human cells due to phosphorylation-dependent formation of the XRCC1 repair complex Dutta, Arijit Eckelmann, Bradley Adhikari, Sanjay Ahmed, Kazi Mokim Sengupta, Shiladitya Pandey, Arvind Hegde, Pavana M. Tsai, Miaw-Sheue Tainer, John A. Weinfeld, Michael Hegde, Muralidhar L. Mitra, Sankar Nucleic Acids Res Genome Integrity, Repair and Replication Microhomology-mediated end joining (MMEJ), an error-prone pathway for DNA double-strand break (DSB) repair, is implicated in genomic rearrangement and oncogenic transformation; however, its contribution to repair of radiation-induced DSBs has not been characterized. We used recircularization of a linearized plasmid with 3΄-P-blocked termini, mimicking those at X-ray-induced strand breaks, to recapitulate DSB repair via MMEJ or nonhomologous end-joining (NHEJ). Sequence analysis of the circularized plasmids allowed measurement of relative activity of MMEJ versus NHEJ. While we predictably observed NHEJ to be the predominant pathway for DSB repair in our assay, MMEJ was significantly enhanced in preirradiated cells, independent of their radiation-induced arrest in the G2/M phase. MMEJ activation was dependent on XRCC1 phosphorylation by casein kinase 2 (CK2), enhancing XRCC1's interaction with the end resection enzymes MRE11 and CtIP. Both endonuclease and exonuclease activities of MRE11 were required for MMEJ, as has been observed for homology-directed DSB repair (HDR). Furthermore, the XRCC1 co-immunoprecipitate complex (IP) displayed MMEJ activity in vitro, which was significantly elevated after irradiation. Our studies thus suggest that radiation-mediated enhancement of MMEJ in cells surviving radiation therapy may contribute to their radioresistance and could be therapeutically targeted. Oxford University Press 2017-03-17 2016-12-19 /pmc/articles/PMC5389627/ /pubmed/27994036 http://dx.doi.org/10.1093/nar/gkw1262 Text en © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research. http://creativecommons.org/licenses/by-nc/4.0/ This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com
spellingShingle Genome Integrity, Repair and Replication
Dutta, Arijit
Eckelmann, Bradley
Adhikari, Sanjay
Ahmed, Kazi Mokim
Sengupta, Shiladitya
Pandey, Arvind
Hegde, Pavana M.
Tsai, Miaw-Sheue
Tainer, John A.
Weinfeld, Michael
Hegde, Muralidhar L.
Mitra, Sankar
Microhomology-mediated end joining is activated in irradiated human cells due to phosphorylation-dependent formation of the XRCC1 repair complex
title Microhomology-mediated end joining is activated in irradiated human cells due to phosphorylation-dependent formation of the XRCC1 repair complex
title_full Microhomology-mediated end joining is activated in irradiated human cells due to phosphorylation-dependent formation of the XRCC1 repair complex
title_fullStr Microhomology-mediated end joining is activated in irradiated human cells due to phosphorylation-dependent formation of the XRCC1 repair complex
title_full_unstemmed Microhomology-mediated end joining is activated in irradiated human cells due to phosphorylation-dependent formation of the XRCC1 repair complex
title_short Microhomology-mediated end joining is activated in irradiated human cells due to phosphorylation-dependent formation of the XRCC1 repair complex
title_sort microhomology-mediated end joining is activated in irradiated human cells due to phosphorylation-dependent formation of the xrcc1 repair complex
topic Genome Integrity, Repair and Replication
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5389627/
https://www.ncbi.nlm.nih.gov/pubmed/27994036
http://dx.doi.org/10.1093/nar/gkw1262
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