A short G1 phase imposes constitutive replication stress and fork remodelling in mouse embryonic stem cells

Embryonic stem cells (ESCs) represent a transient biological state, where pluripotency is coupled with fast proliferation. ESCs display a constitutively active DNA damage response (DDR), but its molecular determinants have remained elusive. Here we show in cultured ESCs and mouse embryos that H2AX p...

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Bibliographic Details
Published in:Nature communications Vol. 7; no. 1; p. 10660
Main Authors: Ahuja, Akshay K., Jodkowska, Karolina, Teloni, Federico, Bizard, Anna H., Zellweger, Ralph, Herrador, Raquel, Ortega, Sagrario, Hickson, Ian D., Altmeyer, Matthias, Mendez, Juan, Lopes, Massimo
Format: Journal Article
Language:English
Published: London Nature Publishing Group UK 15.02.2016
Nature Publishing Group
Nature Portfolio
Subjects:
631/136/2086
631/136/532/2117
631/337/1427/2566
631/80/86
Animals
Ataxia Telangiectasia Mutated Proteins - metabolism
Blastocyst - metabolism
Blotting, Western
Chromatin - metabolism
Chromosomal Proteins, Non-Histone - metabolism
DNA Damage
DNA Replication
DNA, Single-Stranded - metabolism
DNA-Binding Proteins - metabolism
Electrophoresis, Gel, Pulsed-Field
Flow Cytometry
G1 Phase
G1 Phase Cell Cycle Checkpoints
Histones - metabolism
Humanities and Social Sciences
Inactivation
Mice
Microscopy, Confocal
Microscopy, Electron
Microscopy, Fluorescence
Mitosis
Morula - metabolism
Mouse Embryonic Stem Cells - metabolism
multidisciplinary
Phosphorylation
Poly(ADP-ribose) Polymerases - metabolism
Rad51 Recombinase - metabolism
Replication Protein A - metabolism
Science
Science (multidisciplinary)
Stem cells
Tumor Suppressor p53-Binding Protein 1
ISSN:2041-1723, 2041-1723
Online Access:Get full text
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Summary:Embryonic stem cells (ESCs) represent a transient biological state, where pluripotency is coupled with fast proliferation. ESCs display a constitutively active DNA damage response (DDR), but its molecular determinants have remained elusive. Here we show in cultured ESCs and mouse embryos that H2AX phosphorylation is dependent on Ataxia telangiectasia and Rad3 related (ATR) and is associated with chromatin loading of the ssDNA-binding proteins RPA and RAD51. Single-molecule analysis of replication intermediates reveals massive ssDNA gap accumulation, reduced fork speed and frequent fork reversal. All these marks of replication stress do not impair the mitotic process and are rapidly lost at differentiation onset. Delaying the G1/S transition in ESCs allows formation of 53BP1 nuclear bodies and suppresses ssDNA accumulation, fork slowing and reversal in the following S-phase. Genetic inactivation of fork slowing and reversal leads to chromosomal breakage in unperturbed ESCs. We propose that rapid cell cycle progression makes ESCs dependent on effective replication-coupled mechanisms to protect genome integrity. In fast proliferating embryonic stem cells (ESC) the DNA damage response is activated by mechanisms that are as yet elusive. Here, Ahuja et al. link the DNA damage response to replication stress in mouse ESCs, caused by a short G1 phase, and propose fork remodelling as maintaining genome stability in embryos.
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ISSN:2041-1723
2041-1723
DOI:10.1038/ncomms10660