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Updated: Jun 1, 2026
Detection of DNA Double-Stranded Breaks in Mouse Oocytes
Published on: June 23, 2023
DNA damage-sensing kinases mediate the mouse 2-cell embryo's response to genotoxic stress
X F Mu1, X L Jin, M M J Farnham
1Human Reproduction Unit, Sydney Centre for Developmental and Regenerative Medicine, Kolling Institute of Medical Research, Sydney Medical School, University of Sydney, Sydney, New South Wales, Australia.
Abstract:
A critical function of cells is the maintenance of their genomic integrity. A family of phosphoinositide-3-kinase-related protein kinases, which includes ataxia telangiectasia mutated (ATM) and ataxia telangiectasia and Rad3 related (ATR) kinases, play key roles in sensing DNA damage. ATM and ATR were demonstrated in the cleavage stages of mouse embryo development. Genotoxic stress was imposed by exposure to ultraviolet (UV) radiation (causes DNA strand breaks) or cisplatin (causes strand cross-links). UV irradiation or cisplatin treatment of 2-cell embryos in the G(2) phase of the cell cycle caused DNA damage as defined by increased phosphorylation of the H2A histone family, member X (H2AFX; previously H2AX) variant. UV irradiation caused a stable G(2)-M arrest, and cisplatin treatment allowed progression through mitosis followed by activation of a G(1)-S checkpoint. Both checkpoints were transformation-related protein 53-independent. Caffeine (inhibits both ATM and ATR), but not KU55933 (ATM-selective inhibitor), reversed the G(2)-M block induced by UV, inferring a primary role for ATR in sensing this form of DNA damage. Caffeine and KU55933 were equally effective in reversing the cisplatin-induced G(1)-S block, implicating ATM as the primary sensing enzyme. Breaching of either checkpoint by treatment with caffeine or KU55933 allowed embryos to progress through several further cell cycles, yet none developed to blastocysts. The results show, to our knowledge for the first time, that the G(2)-M and G(1)-S cell-cycle checkpoints in the early embryo are differentially regulated by ATM and ATR in response to genotoxic stress and that they act as an initial point for containment of genomic damage. Under conditions of extensive or persistent DNA damage, the demise of the embryo is the ultimate method of protecting genomic integrity.
Insights
Early mouse embryos utilize distinct DNA damage checkpoints, regulated by ATM and ATR kinases, to maintain genomic integrity. These checkpoints prevent development following genotoxic stress, ultimately leading to embryo demise if damage is severe.
Area of Science:
- Cell Biology
- Developmental Biology
- Genetics
Background:
- Genomic integrity is crucial for cellular function and organismal development.
- Phosphoinositide-3-kinase-related kinases, including ATM and ATR, are key sensors of DNA damage.
- ATM and ATR kinases are present during early mouse embryonic development.
Purpose of the Study:
- To investigate the roles of ATM and ATR in regulating cell-cycle checkpoints in response to genotoxic stress during early mouse embryonic development.
- To determine how UV radiation and cisplatin treatment differentially affect cell-cycle progression and checkpoint activation in 2-cell mouse embryos.
- To elucidate the specific contributions of ATM and ATR to the G(2)-M and G(1)-S checkpoints.
Main Methods:
- Exposure of 2-cell mouse embryos to genotoxic agents: ultraviolet (UV) radiation and cisplatin.
- Assessment of DNA damage via phosphorylation of H2AFX.
- Pharmacological inhibition of ATM and ATR using caffeine and KU55933 to analyze checkpoint recovery.
- Monitoring of cell-cycle progression and embryonic development to the blastocyst stage.
Main Results:
- UV irradiation induced DNA damage and a G(2)-M cell-cycle arrest, primarily mediated by ATR.
- Cisplatin treatment caused DNA damage and a G(1)-S checkpoint activation, primarily mediated by ATM.
- Both checkpoints were independent of p53.
- Inhibition of these checkpoints allowed further cell cycles but prevented blastocyst development.
- Embryo demise was observed under conditions of extensive or persistent DNA damage.
Conclusions:
- The G(2)-M and G(1)-S cell-cycle checkpoints in early embryos are differentially regulated by ATM and ATR in response to genotoxic stress.
- These checkpoints serve as an initial mechanism for containing genomic damage during early development.
- Severe or persistent DNA damage leads to embryo demise as a mechanism to protect genomic integrity.
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