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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...

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Autores principales: Rassoolzadeh, Hanif, Coucoravas, Christos, Farnebo, Marianne
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Taylor & Francis 2016
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.
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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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