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[Analysis of irradiation-induced repair foci in mouse embryonic stem cells]
Abstract:
Somatic cells in response to DNA damage activate two important protective mechanisms: G1 checkpoint control and a program for recognizing and repairing DNA defects (DDR signaling). Both mechanisms are triggered by the activation of common sensor kinases ATM and ATR, which in turn phosphorylate downstream targets. Mouse embryonic stem cells (mESCs) lack of G1 checkpoint and undergo only temporary G2 delay after DNA damage. We have analyzed the ability of mESCs to detect DNA damage and to form repair foci after irradiation. We showed irradiation-induced activation of ATM and ATR is followed by formation of γH2AX foci co-localized with DNA repair proteins Rad51, DNA-PK and adapter protein 53BP1. Furthermore, we checked contribution of ATM/Chk2 and ATR/Chk1 cascades to cell cycle control and viability of mESCs after DNA damage. Inhibition of ATR/Chk1 cascade leads to accumulation of G1 phase cells, whereas perturbation of ATM/Chk2 activity causes no such effect. Moreover, inhibition of ATR/Chk1 activity, but not ATM/Chk2, substantially augments the killing effect of ionizing radiation on mESCs. In summary, our results indicate that mESCs are capable of recognizing DNA damage and forming repair foci, but their DDR signaling it seems to be distinct from somatic cells and tightly connected with maintaining of pluripotency and self-renewal.
Insights
Mouse embryonic stem cells (mESCs) detect DNA damage and form repair foci. However, their DNA damage response (DDR) signaling differs from somatic cells, impacting pluripotency and self-renewal.
Area of Science:
- Cell Biology
- Molecular Biology
- Genetics
Background:
- Somatic cells utilize G1 checkpoint control and DNA damage response (DDR) signaling for DNA repair.
- ATM and ATR kinases are key sensors in DNA damage response pathways.
- Mouse embryonic stem cells (mESCs) exhibit a distinct cell cycle response to DNA damage, lacking G1 checkpoint control.
Purpose of the Study:
- To investigate the DNA damage detection and repair foci formation capabilities of mESCs.
- To analyze the roles of ATM/Chk2 and ATR/Chk1 signaling cascades in mESC cell cycle control and viability post-irradiation.
- To understand the unique DDR signaling mechanisms in mESCs compared to somatic cells.
Main Methods:
- Irradiation of mESCs to induce DNA damage.
- Analysis of DNA repair foci formation (γH2AX, Rad51, DNA-PK, 53BP1).
- Inhibition of ATM/Chk2 and ATR/Chk1 signaling pathways to assess their impact on cell cycle and viability.
Main Results:
- Irradiation induced ATM and ATR activation, leading to the formation of γH2AX foci co-localized with DNA repair proteins.
- Inhibition of ATR/Chk1, but not ATM/Chk2, caused G1 phase cell accumulation.
- ATR/Chk1 inhibition significantly increased mESC sensitivity to ionizing radiation, unlike ATM/Chk2 inhibition.
Conclusions:
- mESCs can recognize DNA damage and form repair foci.
- mESC DDR signaling is distinct from somatic cells and linked to maintaining pluripotency and self-renewal.
- The ATR/Chk1 pathway plays a crucial role in mESC survival following DNA damage.
