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

Akshay K Ahuja,Juan Mendez,Federico Teloni,Karolina Jodkowska,Ralph Zellweger,Matthias Altmeyer,Sagrario Ortega,Raquel Herrador,Massimo Lopes,Anna H Bizard,Ian D Hickson

doi:10.1038/ncomms10660

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.

Highlights

Embryonic stem cells (ESCs) represent a transient biological state, where pluripotency is coupled with fast proliferation
To shed light on the molecular determinants of gH2AX formation in ESCs, we tested whether other markers of genotoxic stress are detectable in these cells
Unless irradiated, ESCs are devoid of 53BP1 foci (Supplementary Fig. 1a)[9], and found that gH2AX in unperturbed ESCs is invariably associated with extensive chromatin loading of RPA32 and RAD51, two single-stranded DNA-binding proteins involved in recombinational mechanisms at doublestrand break (DSB) and stalled forks[22]

Summary

Introduction

Embryonic stem cells (ESCs) represent a transient biological state, where pluripotency is coupled with fast proliferation. Asynchronously growing ESCs have remarkably short gap phases and spend most of their time in the S-phase, the time spent for genome duplication is not significantly different from that in somatic cells[12] In line with their high proliferative capacity, most positive cell cycle regulators and DNA replication factors (for example, CDC25A, CDC6, cyclins and so on) are extremely abundant in ESCs compared with mouse embryonic fibroblasts (MEFs)[13] and their levels drastically drop down on ESC differentiation[14]. This unusual cell cycle control is orchestrated by key stem cell factors[5,15] and was shown to be essential to maintain pluripotency in ESCs16,17. As hyperproliferation and replication problems in adult stem cells have been recently linked to cancer onset and stem cell attrition[20,21], molecular mechanisms related to those described here in early embryogenesis may underlie key causative events in human disease

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Conclusion