Pathways of repair of DNA double strand breaks (DSBs) cooperate with DNA damage cell cycle checkpoints to safeguard genomic stability when cells are exposed to ionizing radiation (IR). It is widely accepted that checkpoints facilitate the function of DSB repair pathways. Whether DSB repair proficiency feeds back into checkpoint activation is less well investigated. Here, we study activation of the G₂-checkpoint in cells deficient in homologous recombination repair (HRR) after exposure to low IR doses (∼1 Gy) in the G₂-phase. We report that in the absence of functional HRR, activation of the G₂-checkpoint is severely impaired. This response is specific for HRR, as cells deficient in classical non-homologous end joining (c-NHEJ) develop a similar or stronger G₂-checkpoint than wild-type (WT) cells. Inhibition of ATM or ATR leaves largely unaffected residual G₂-checkpoint in HRR-deficient cells, suggesting that the G₂-checkpoint engagement of ATM/ATR is coupled to HRR. HRR-deficient cells show in G₂-phase reduced DSB-end-resection, as compared to WT-cells or c-NHEJ mutants, confirming the reported link between resection and G₂-checkpoint activation. Strikingly, at higher IR doses (≥4 Gy) HRR-deficient cells irradiated in G₂-phase activate a weak but readily detectable ATM/ATR-dependent G₂-checkpoint, whereas HRR-deficient cells irradiated in S-phase develop a stronger G₂-checkpoint than WT-cells. We conclude that HRR and the ATM/ATR-dependent G₂-checkpoint are closely intertwined in cells exposed to low IR-doses in G₂-phase, where HRR dominates; they uncouple as HRR becomes suppressed at higher IR doses. Notably, this coupling is specific for cells irradiated in G₂-phase, and cells irradiated in S-phase utilize a different mechanistic setup.

当细胞暴露于电离辐射 (IR) 时，DNA 双链断裂 (DSB) 修复途径与 DNA 损伤细胞周期检查点合作，以保护基因组稳定性。人们普遍认为，检查点有助于 DSB 修复途径的功能。DSB 修复熟练程度是否会反馈到检查点激活中还没有得到很好的研究。在这里，我们研究的G活化₂在暴露于在G低IR剂量（〜1戈瑞）后同源重组修复（HRR）缺陷细胞检控点₂ -PHASE。我们报告说，在没有功能性 HRR 的情况下，G ₂检查点的激活受到严重损害。这种反应对 HRR 是特异性的，因为在经典的非同源末端连接 (c-NHEJ) 中存在缺陷的细胞会产生类似或更强的 G_2-检查点比野生型 (WT) 细胞高。ATM 或 ATR 的抑制使HRR 缺陷细胞中的残留 G _{2 -}检查点基本上不受影响，这表明ATM/ATR的 G _{2 -}检查点参与与 HRR 耦合。与 WT 细胞或 c-NHEJ 突变体相比，HRR 缺陷细胞在 G _{2 期}显示减少的 DSB 末端切除，证实了切除和 G ₂检查点激活之间的报告联系。引人注目的是，在较高的 IR 剂量（≥4 Gy）下，在 G _{2 期}照射的 HRR 缺陷细胞激活了一个微弱但易于检测的 ATM/ATR 依赖性 G ₂检查点，而在 S 期照射的 HRR 缺陷细胞则产生更强的摹₂-检查点比 WT 细胞。我们得出结论，HRR 和依赖于 ATM/ATR 的 G ₂检查点在 G _{2 期}暴露于低 IR 剂量的细胞中紧密交织在一起，其中 HRR 占主导地位；当 HRR 在更高的 IR 剂量下被抑制时，它们会解耦。值得注意的是，这种耦合对在 G _{2 期}照射的细胞具有特异性，在 S 期照射的细胞利用不同的机制设置。