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The proximity ligation assay reveals that at DNA double-strand breaks WRAP53β associates with γH2AX and controls interactions between RNF8 and MDC1
We recently demonstrated that WRAP53β acts as a key regulator of ubiquitin-dependent repair of DNA double-strand breaks. Here, we applied the proximity ligation assay (PLA) to show that at such breaks WRAP53β accumulates in close proximity to γH2AX and, furthermore as demonstrated by their co-immuno...
Autores principales: | , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Taylor & Francis
2016
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4915514/ https://www.ncbi.nlm.nih.gov/pubmed/26734725 http://dx.doi.org/10.1080/19491034.2015.1106675 |
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author | Rassoolzadeh, Hanif Coucoravas, Christos Farnebo, Marianne |
author_facet | Rassoolzadeh, Hanif Coucoravas, Christos Farnebo, Marianne |
author_sort | Rassoolzadeh, Hanif |
collection | PubMed |
description | We recently demonstrated that WRAP53β acts as a key regulator of ubiquitin-dependent repair of DNA double-strand breaks. Here, we applied the proximity ligation assay (PLA) to show that at such breaks WRAP53β accumulates in close proximity to γH2AX and, furthermore as demonstrated by their co-immunoprecipitation (IP) binds to γH2AX, in a manner dependent on the ATM and ATR kinases. Moreover, formation of complexes between MDC1 and both its partners RNF8 and phosphorylated ATM was visualized. The interaction of MDC1 with RNF8, but not with ATM requires WRAP53β, suggesting that WRAP53β facilitates the former interaction without altering phosphorylation of MDC1 by ATM. Furthermore, our findings highlight PLA as a more sensitive method for the analysis of recruitment of repair factors and complex formation at DNA breaks that are difficult to detect using conventional immunofluorescence. |
format | Online Article Text |
id | pubmed-4915514 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2016 |
publisher | Taylor & Francis |
record_format | MEDLINE/PubMed |
spelling | pubmed-49155142016-07-06 The proximity ligation assay reveals that at DNA double-strand breaks WRAP53β associates with γH2AX and controls interactions between RNF8 and MDC1 Rassoolzadeh, Hanif Coucoravas, Christos Farnebo, Marianne Nucleus Report We recently demonstrated that WRAP53β acts as a key regulator of ubiquitin-dependent repair of DNA double-strand breaks. Here, we applied the proximity ligation assay (PLA) to show that at such breaks WRAP53β accumulates in close proximity to γH2AX and, furthermore as demonstrated by their co-immunoprecipitation (IP) binds to γH2AX, in a manner dependent on the ATM and ATR kinases. Moreover, formation of complexes between MDC1 and both its partners RNF8 and phosphorylated ATM was visualized. The interaction of MDC1 with RNF8, but not with ATM requires WRAP53β, suggesting that WRAP53β facilitates the former interaction without altering phosphorylation of MDC1 by ATM. Furthermore, our findings highlight PLA as a more sensitive method for the analysis of recruitment of repair factors and complex formation at DNA breaks that are difficult to detect using conventional immunofluorescence. Taylor & Francis 2016-01-06 /pmc/articles/PMC4915514/ /pubmed/26734725 http://dx.doi.org/10.1080/19491034.2015.1106675 Text en © 2015 The Author(s). Published with license by Taylor & Francis Group, LLC http://creativecommons.org/licenses/by-nc/3.0/ This is an Open Access article distributed under the terms of the Creative Commons Attribution-Non-Commercial License http://creativecommons.org/licenses/by-nc/3.0/, which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. The moral rights of the named author(s) have been asserted. |
spellingShingle | Report Rassoolzadeh, Hanif Coucoravas, Christos Farnebo, Marianne The proximity ligation assay reveals that at DNA double-strand breaks WRAP53β associates with γH2AX and controls interactions between RNF8 and MDC1 |
title | The proximity ligation assay reveals that at DNA double-strand breaks WRAP53β associates with γH2AX and controls interactions between RNF8 and MDC1 |
title_full | The proximity ligation assay reveals that at DNA double-strand breaks WRAP53β associates with γH2AX and controls interactions between RNF8 and MDC1 |
title_fullStr | The proximity ligation assay reveals that at DNA double-strand breaks WRAP53β associates with γH2AX and controls interactions between RNF8 and MDC1 |
title_full_unstemmed | The proximity ligation assay reveals that at DNA double-strand breaks WRAP53β associates with γH2AX and controls interactions between RNF8 and MDC1 |
title_short | The proximity ligation assay reveals that at DNA double-strand breaks WRAP53β associates with γH2AX and controls interactions between RNF8 and MDC1 |
title_sort | proximity ligation assay reveals that at dna double-strand breaks wrap53β associates with γh2ax and controls interactions between rnf8 and mdc1 |
topic | Report |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4915514/ https://www.ncbi.nlm.nih.gov/pubmed/26734725 http://dx.doi.org/10.1080/19491034.2015.1106675 |
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