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Separable roles for Mec1/ATR in genome maintenance, DNA replication, and checkpoint signaling

The Mec1/ATR kinase coordinates multiple cellular responses to replication stress. In addition to its canonical role in activating the checkpoint kinase Rad53, Mec1 also plays checkpoint-independent roles in genome maintenance that are not well understood. Here we used a combined genetic–phosphoprot...

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Autores principales: Lanz, Michael Charles, Oberly, Susannah, Sanford, Ethan James, Sharma, Sushma, Chabes, Andrei, Smolka, Marcus Bustamante
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Cold Spring Harbor Laboratory Press 2018
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6049512/
https://www.ncbi.nlm.nih.gov/pubmed/29899143
http://dx.doi.org/10.1101/gad.308148.117
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author Lanz, Michael Charles
Oberly, Susannah
Sanford, Ethan James
Sharma, Sushma
Chabes, Andrei
Smolka, Marcus Bustamante
author_facet Lanz, Michael Charles
Oberly, Susannah
Sanford, Ethan James
Sharma, Sushma
Chabes, Andrei
Smolka, Marcus Bustamante
author_sort Lanz, Michael Charles
collection PubMed
description The Mec1/ATR kinase coordinates multiple cellular responses to replication stress. In addition to its canonical role in activating the checkpoint kinase Rad53, Mec1 also plays checkpoint-independent roles in genome maintenance that are not well understood. Here we used a combined genetic–phosphoproteomic approach to manipulate Mec1 activation and globally monitor Mec1 signaling, allowing us to delineate distinct checkpoint-independent modes of Mec1 action. Using cells in which endogenous Mec1 activators were genetically ablated, we found that expression of “free” Mec1 activation domains (MADs) can robustly activate Mec1 and rescue the severe DNA replication and growth defects of these cells back to wild-type levels. However, unlike the activation mediated by endogenous activator proteins, “free” MADs are unable to stimulate Mec1-mediated suppression of gross chromosomal rearrangements (GCRs), revealing that Mec1's role in genome maintenance is separable from a previously unappreciated proreplicative function. Both Mec1's functions in promoting replication and suppressing GCRs are independent of the downstream checkpoint kinases. Additionally, Mec1-dependent GCR suppression seems to require localized Mec1 action at DNA lesions, which correlates with the phosphorylation of activator-proximal substrates involved in homologous recombination-mediated DNA repair. These findings establish that Mec1 initiates checkpoint signaling, promotes DNA replication, and maintains genetic stability through distinct modes of action.
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spelling pubmed-60495122018-12-01 Separable roles for Mec1/ATR in genome maintenance, DNA replication, and checkpoint signaling Lanz, Michael Charles Oberly, Susannah Sanford, Ethan James Sharma, Sushma Chabes, Andrei Smolka, Marcus Bustamante Genes Dev Research Paper The Mec1/ATR kinase coordinates multiple cellular responses to replication stress. In addition to its canonical role in activating the checkpoint kinase Rad53, Mec1 also plays checkpoint-independent roles in genome maintenance that are not well understood. Here we used a combined genetic–phosphoproteomic approach to manipulate Mec1 activation and globally monitor Mec1 signaling, allowing us to delineate distinct checkpoint-independent modes of Mec1 action. Using cells in which endogenous Mec1 activators were genetically ablated, we found that expression of “free” Mec1 activation domains (MADs) can robustly activate Mec1 and rescue the severe DNA replication and growth defects of these cells back to wild-type levels. However, unlike the activation mediated by endogenous activator proteins, “free” MADs are unable to stimulate Mec1-mediated suppression of gross chromosomal rearrangements (GCRs), revealing that Mec1's role in genome maintenance is separable from a previously unappreciated proreplicative function. Both Mec1's functions in promoting replication and suppressing GCRs are independent of the downstream checkpoint kinases. Additionally, Mec1-dependent GCR suppression seems to require localized Mec1 action at DNA lesions, which correlates with the phosphorylation of activator-proximal substrates involved in homologous recombination-mediated DNA repair. These findings establish that Mec1 initiates checkpoint signaling, promotes DNA replication, and maintains genetic stability through distinct modes of action. Cold Spring Harbor Laboratory Press 2018-06-01 /pmc/articles/PMC6049512/ /pubmed/29899143 http://dx.doi.org/10.1101/gad.308148.117 Text en © 2018 Lanz et al.; Published by Cold Spring Harbor Laboratory Press http://creativecommons.org/licenses/by-nc/4.0/ This article is distributed exclusively by Cold Spring Harbor Laboratory Press for the first six months after the full-issue publication date (see http://genesdev.cshlp.org/site/misc/terms.xhtml). After six months, it is available under a Creative Commons License (Attribution-NonCommercial 4.0 International), as described at http://creativecommons.org/licenses/by-nc/4.0/.
spellingShingle Research Paper
Lanz, Michael Charles
Oberly, Susannah
Sanford, Ethan James
Sharma, Sushma
Chabes, Andrei
Smolka, Marcus Bustamante
Separable roles for Mec1/ATR in genome maintenance, DNA replication, and checkpoint signaling
title Separable roles for Mec1/ATR in genome maintenance, DNA replication, and checkpoint signaling
title_full Separable roles for Mec1/ATR in genome maintenance, DNA replication, and checkpoint signaling
title_fullStr Separable roles for Mec1/ATR in genome maintenance, DNA replication, and checkpoint signaling
title_full_unstemmed Separable roles for Mec1/ATR in genome maintenance, DNA replication, and checkpoint signaling
title_short Separable roles for Mec1/ATR in genome maintenance, DNA replication, and checkpoint signaling
title_sort separable roles for mec1/atr in genome maintenance, dna replication, and checkpoint signaling
topic Research Paper
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6049512/
https://www.ncbi.nlm.nih.gov/pubmed/29899143
http://dx.doi.org/10.1101/gad.308148.117
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