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...
| Autores: | , , , , , , , , , , |
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| Tipo de recurso: | artículo |
| Fecha de publicación: | 2016 |
| País: | España |
| Institución: | Instituto de Salud Carlos III (ISCIII) |
| Repositorio: | Repisalud |
| Idioma: | inglés |
| OAI Identifier: | oai:repisalud.isciii.es:20.500.12105/7866 |
| Acceso en línea: | http://hdl.handle.net/20.500.12105/7866 |
| Access Level: | acceso abierto |
| Palabra clave: | Animals Ataxia Telangiectasia Mutated Proteins Blastocyst Blotting, Western Chromatin Chromosomal Proteins, Non-Histone DNA, Single-Stranded DNA-Binding Proteins Electrophoresis, Gel, Pulsed-Field Flow Cytometry Histones Mice Microscopy, Confocal Microscopy, Electron Microscopy, Fluorescence Mitosis Morula Mouse Embryonic Stem Cells Phosphorylation Poly(ADP-ribose) Polymerases Rad51 Recombinase Replication Protein A Tumor Suppressor p53-Binding Protein 1 DNA Damage DNA Replication G1 Phase G1 Phase Cell Cycle Checkpoints |
| Sumario: | 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. |
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