title: A short G1 phase imposes constitutive replication stress and fork remodelling in mouse embryonic stem cells.
abstract: 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.
authors.affiliation: Institute of Molecular Cancer Research, University of Zurich, Zurich CH-8057, Switzerland.
authors.forename: Akshay K
authors.initials: AK
authors.lastname: Ahuja
authors.affiliation: DNA Replication Group, Molecular Oncology Programme, CNIO, Madrid E-28029, Spain.
authors.forename: Karolina
authors.initials: K
authors.lastname: Jodkowska
authors.affiliation: Institute of Veterinary Biochemistry and Molecular Biology, University of Zurich, Zurich CH-8057, Switzerland.
authors.forename: Federico
authors.identifier: http://orcid.org/0000-0003-0953-8010
authors.initials: F
authors.lastname: Teloni
authors.affiliation: Department of Cellular and Molecular Medicine, Center for Chromosome Stability and Center for Healthy Aging, University of Copenhagen, Panum Institute, Copenhagen N DK-2200, Denmark.
authors.forename: Anna H
authors.identifier: http://orcid.org/0000-0003-1858-3999
authors.initials: AH
authors.lastname: Bizard
authors.affiliation: Institute of Molecular Cancer Research, University of Zurich, Zurich CH-8057, Switzerland.
authors.forename: Ralph
authors.initials: R
authors.lastname: Zellweger
authors.affiliation: Institute of Molecular Cancer Research, University of Zurich, Zurich CH-8057, Switzerland.
authors.forename: Raquel
authors.initials: R
authors.lastname: Herrador
authors.affiliation: Transgenic Mice Core Unit, Biotechnology Programme, CNIO, Madrid E-28029, Spain.
authors.forename: Sagrario
authors.initials: S
authors.lastname: Ortega
authors.affiliation: Department of Cellular and Molecular Medicine, Center for Chromosome Stability and Center for Healthy Aging, University of Copenhagen, Panum Institute, Copenhagen N DK-2200, Denmark.
authors.forename: Ian D
authors.initials: ID
authors.lastname: Hickson
authors.affiliation: Institute of Veterinary Biochemistry and Molecular Biology, University of Zurich, Zurich CH-8057, Switzerland.
authors.forename: Matthias
authors.initials: M
authors.lastname: Altmeyer
authors.affiliation: DNA Replication Group, Molecular Oncology Programme, CNIO, Madrid E-28029, Spain.
authors.forename: Juan
authors.initials: J
authors.lastname: Mendez
authors.affiliation: Institute of Molecular Cancer Research, University of Zurich, Zurich CH-8057, Switzerland.
authors.forename: Massimo
authors.initials: M
authors.lastname: Lopes
mesh_terms: D000818:Animals; D064007:Ataxia Telangiectasia Mutated Proteins / Q000378:metabolism; D001755:Blastocyst / Q000378:metabolism; D015153:Blotting, Western; D002843:Chromatin / Q000378:metabolism; D002868:Chromosomal Proteins, Non-Histone / Q000378:metabolism; D004249:DNA Damage; D004261:DNA Replication; D004277:DNA, Single-Stranded / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism*; D016521:Electrophoresis, Gel, Pulsed-Field; D005434:Flow Cytometry; D016193:G1 Phase; D059585:G1 Phase Cell Cycle Checkpoints; D006657:Histones / Q000378:metabolism; D051379:Mice; D018613:Microscopy, Confocal; D008854:Microscopy, Electron; D008856:Microscopy, Fluorescence; D008938:Mitosis; D009028:Morula / Q000378:metabolism; D000066450:Mouse Embryonic Stem Cells / Q000378:metabolism*; D010766:Phosphorylation; D011065:Poly(ADP-ribose) Polymerases / Q000378:metabolism; D051135:Rad51 Recombinase / Q000378:metabolism*; D051716:Replication Protein A / Q000378:metabolism; D000071857:Tumor Suppressor p53-Binding Protein 1
chemical_list: D064007:Ataxia Telangiectasia Mutated Proteins; D002843:Chromatin; D002868:Chromosomal Proteins, Non-Histone; D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; D006657:Histones; D011065:Poly(ADP-ribose) Polymerases; D051135:Rad51 Recombinase; D051716:Replication Protein A; D000071857:Tumor Suppressor p53-Binding Protein 1; C000608616:RPA32 protein, mouse; C498302:Trp53bp1 protein, mouse; C576897:Atr protein, mouse; C495154:Rad51 protein, mouse; C475961:gamma-H2AX protein, mouse