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Structure-function analysis of TOPBP1’s role in ATR signaling using the DSB-mediated ATR activation in Xenopus egg extracts (DMAX) system
The protein kinase ATR is activated at sites of DNA double-strand breaks where it plays important roles in promoting DNA end resection and regulating cell cycle progression. TOPBP1 is a multi BRCT repeat containing protein that activates ATR at DSBs. Here we have developed an experimental tool, the...
Autores principales: | , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Nature Publishing Group UK
2021
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7801695/ https://www.ncbi.nlm.nih.gov/pubmed/33432091 http://dx.doi.org/10.1038/s41598-020-80626-1 |
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author | Montales, Katrina Kim, Ahhyun Ruis, Kenna Michael, W. Matthew |
author_facet | Montales, Katrina Kim, Ahhyun Ruis, Kenna Michael, W. Matthew |
author_sort | Montales, Katrina |
collection | PubMed |
description | The protein kinase ATR is activated at sites of DNA double-strand breaks where it plays important roles in promoting DNA end resection and regulating cell cycle progression. TOPBP1 is a multi BRCT repeat containing protein that activates ATR at DSBs. Here we have developed an experimental tool, the DMAX system, to study the biochemical mechanism for TOPBP1-mediated ATR signalling. DMAX combines simple, linear dsDNA molecules with Xenopus egg extracts and results in a physiologically relevant, DSB-induced activation of ATR. We find that DNAs of 5000 nucleotides, at femtomolar concentration, potently activate ATR in this system. By combining immunodepletion and add-back of TOPBP1 point mutants we use DMAX to determine which of TOPBP1’s nine BRCT domains are required for recruitment of TOPBP1 to DSBs and which domains are needed for ATR-mediated phosphorylation of CHK1. We find that BRCT1 and BRCT7 are important for recruitment and that BRCT5 functions downstream of recruitment to promote ATR-mediated phosphorylation of CHK1. We also show that BRCT7 plays a second role, independent of recruitment, in promoting ATR signalling. These findings supply a new research tool for, and new insights into, ATR biology. |
format | Online Article Text |
id | pubmed-7801695 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2021 |
publisher | Nature Publishing Group UK |
record_format | MEDLINE/PubMed |
spelling | pubmed-78016952021-01-13 Structure-function analysis of TOPBP1’s role in ATR signaling using the DSB-mediated ATR activation in Xenopus egg extracts (DMAX) system Montales, Katrina Kim, Ahhyun Ruis, Kenna Michael, W. Matthew Sci Rep Article The protein kinase ATR is activated at sites of DNA double-strand breaks where it plays important roles in promoting DNA end resection and regulating cell cycle progression. TOPBP1 is a multi BRCT repeat containing protein that activates ATR at DSBs. Here we have developed an experimental tool, the DMAX system, to study the biochemical mechanism for TOPBP1-mediated ATR signalling. DMAX combines simple, linear dsDNA molecules with Xenopus egg extracts and results in a physiologically relevant, DSB-induced activation of ATR. We find that DNAs of 5000 nucleotides, at femtomolar concentration, potently activate ATR in this system. By combining immunodepletion and add-back of TOPBP1 point mutants we use DMAX to determine which of TOPBP1’s nine BRCT domains are required for recruitment of TOPBP1 to DSBs and which domains are needed for ATR-mediated phosphorylation of CHK1. We find that BRCT1 and BRCT7 are important for recruitment and that BRCT5 functions downstream of recruitment to promote ATR-mediated phosphorylation of CHK1. We also show that BRCT7 plays a second role, independent of recruitment, in promoting ATR signalling. These findings supply a new research tool for, and new insights into, ATR biology. Nature Publishing Group UK 2021-01-11 /pmc/articles/PMC7801695/ /pubmed/33432091 http://dx.doi.org/10.1038/s41598-020-80626-1 Text en © The Author(s) 2021 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. |
spellingShingle | Article Montales, Katrina Kim, Ahhyun Ruis, Kenna Michael, W. Matthew Structure-function analysis of TOPBP1’s role in ATR signaling using the DSB-mediated ATR activation in Xenopus egg extracts (DMAX) system |
title | Structure-function analysis of TOPBP1’s role in ATR signaling using the DSB-mediated ATR activation in Xenopus egg extracts (DMAX) system |
title_full | Structure-function analysis of TOPBP1’s role in ATR signaling using the DSB-mediated ATR activation in Xenopus egg extracts (DMAX) system |
title_fullStr | Structure-function analysis of TOPBP1’s role in ATR signaling using the DSB-mediated ATR activation in Xenopus egg extracts (DMAX) system |
title_full_unstemmed | Structure-function analysis of TOPBP1’s role in ATR signaling using the DSB-mediated ATR activation in Xenopus egg extracts (DMAX) system |
title_short | Structure-function analysis of TOPBP1’s role in ATR signaling using the DSB-mediated ATR activation in Xenopus egg extracts (DMAX) system |
title_sort | structure-function analysis of topbp1’s role in atr signaling using the dsb-mediated atr activation in xenopus egg extracts (dmax) system |
topic | Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7801695/ https://www.ncbi.nlm.nih.gov/pubmed/33432091 http://dx.doi.org/10.1038/s41598-020-80626-1 |
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