Structure-function analysis of TOPBP1’s role in ATR signaling using the DSB-mediated ATR activation in Xenopus egg extracts (DMAX) system

Abstract 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 t...

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Autores principales:	Katrina Montales, Ahhyun Kim, Kenna Ruis, W. Matthew Michael
Formato:	article
Lenguaje:	EN
Publicado:	Nature Portfolio 2021
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/8c802708d8794d5294da91ea9152b9c8
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spelling	oai:doaj.org-article:8c802708d8794d5294da91ea9152b9c82021-12-02T14:01:22ZStructure-function analysis of TOPBP1’s role in ATR signaling using the DSB-mediated ATR activation in Xenopus egg extracts (DMAX) system10.1038/s41598-020-80626-12045-2322https://doaj.org/article/8c802708d8794d5294da91ea9152b9c82021-01-01T00:00:00Zhttps://doi.org/10.1038/s41598-020-80626-1https://doaj.org/toc/2045-2322Abstract 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.Katrina MontalesAhhyun KimKenna RuisW. Matthew MichaelNature PortfolioarticleMedicineRScienceQENScientific Reports, Vol 11, Iss 1, Pp 1-13 (2021)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Katrina Montales Ahhyun Kim Kenna Ruis W. Matthew Michael Structure-function analysis of TOPBP1’s role in ATR signaling using the DSB-mediated ATR activation in Xenopus egg extracts (DMAX) system
description	Abstract 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	article
author	Katrina Montales Ahhyun Kim Kenna Ruis W. Matthew Michael
author_facet	Katrina Montales Ahhyun Kim Kenna Ruis W. Matthew Michael
author_sort	Katrina Montales
title	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_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_sort	structure-function analysis of topbp1’s role in atr signaling using the dsb-mediated atr activation in xenopus egg extracts (dmax) system
publisher	Nature Portfolio
publishDate	2021
url	https://doaj.org/article/8c802708d8794d5294da91ea9152b9c8
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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

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