Suvorova I I, Pospelov V A
Tsitologiia. 2014;56(5):340-5.
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.
体细胞在响应DNA损伤时会激活两种重要的保护机制：G1期检查点控制以及识别和修复DNA缺陷的程序（DNA损伤应答信号传导）。这两种机制均由常见的传感激酶ATM和ATR的激活触发，进而使下游靶点磷酸化。小鼠胚胎干细胞（mESC）缺乏G1期检查点，在DNA损伤后仅经历短暂的G2期延迟。我们分析了mESC检测DNA损伤以及在照射后形成修复灶的能力。我们发现，照射诱导的ATM和ATR激活之后，会形成与DNA修复蛋白Rad51、DNA-PK和衔接蛋白53BP1共定位的γH2AX灶。此外，我们检查了ATM/Chk2和ATR/Chk1级联反应对DNA损伤后mESC细胞周期控制和活力的作用。抑制ATR/Chk1级联反应会导致G1期细胞积累，而干扰ATM/Chk2活性则不会产生这种效果。此外，抑制ATR/Chk1活性而非ATM/Chk2活性，会显著增强电离辐射对mESC的杀伤作用。总之，我们的结果表明，mESC能够识别DNA损伤并形成修复灶，但其DNA损伤应答信号传导似乎与体细胞不同，且与多能性和自我更新的维持紧密相关。
