The cell-killing effect of radiotherapy largely depends on unrepaired DNA double-stranded breaks (DSBs) or lethal chromosome aberrations induced by DSBs. Thus, the capability of DSB repair is critically important for the cancer-cell-killing effect of ionizing radiation. Here, we investigated the involvement of the DNA damage signaling factors ataxia telangiectasia mutated (ATM), ring finger protein 8 (RNF8), and RNF168 in quiescent G0/G1 cells, which are expressed in the majority of cell populations in tumors, after high linear energy transfer (LET) carbon-ion irradiation. Interestingly, ATM inhibition caused a substantial DSB repair defect after high-LET carbon-ion irradiation. Similarly, RNF8 or RNF168 depletion caused a substantial DSB repair defect. ATM inhibition did not exert an additive effect in RNF8-depleted cells, suggesting that ATM and RNF8 function in the same pathway. Importantly, we found that the RNF8 RING mutant showed a similar DSB repair defect, suggesting the requirement of ubiquitin ligase activity in this repair pathway. The RNF8 FHA domain was also required for DSB repair in this axis. Furthermore, the p53-binding protein 1 (53BP1), which is an important downstream factor in RNF8-dependent DSB repair, was also required for this repair. Importantly, either ATM inhibition or RNF8 depletion increased the frequency of chromosomal breaks, but reduced dicentric chromosome formation, demonstrating that ATM/RNF8 is required for the rejoining of DSB ends for the formation of dicentric chromosomes. Finally, we showed that RNF8 depletion augmented radiosensitivity after high-LET carbon-ion irradiation. This study suggests that the inhibition of RNF8 activity or its downstream pathway may augment the efficacy of high-LET carbon-ion therapy.

放射疗法对细胞的杀伤作用在很大程度上取决于未修复的DNA双链断裂（DSB）或由DSB诱导的致死染色体畸变。因此，DSB修复的能力对于电离辐射的癌细胞杀伤作用至关重要。在这里，我们研究了静息的G0 / G1细胞中DNA损伤信号转导因子共济失调毛细血管扩张突变（ATM），无名指蛋白8（RNF8）和RNF168的参与，这些细胞在肿瘤高表达后在大多数细胞群体中均表达线性能量转移（LET）碳离子辐照。有趣的是，在高LET碳离子辐照后，ATM抑制作用导致了实质性的DSB修复缺陷。同样，RNF8或RNF168的耗尽导致了严重的DSB修复缺陷。ATM抑制作用在耗尽RNF8的细胞中未发挥累加作用，提示ATM和RNF8在同一路径中起作用。重要的是，我们发现RNF8 RING突变体显示出类似的DSB修复缺陷，表明在该修复途径中需要泛素连接酶活性。此轴上的DSB修复也需要RNF8 FHA域。此外，该修复还需要p53结合蛋白1（53BP1），它是依赖RNF8的DSB修复的重要下游因素。重要的是，ATM抑制或RNF8耗竭增加了染色体断裂的频率，但减少了双中心染色体的形成，这表明ATM / RNF8是DSB末端重新结合所必需的，以形成双中心染色体。最后，我们显示了高LET碳离子辐照后，RNF8耗竭增强了放射敏感性。