Rothkamm Kai, Krüger Ines, Thompson Larry H, Löbrich Markus
Fachrichtung Biophysik, Universität des Saarlandes, D-66421 Homburg/Saar, Germany.
Mol Cell Biol. 2003 Aug;23(16):5706-15. doi: 10.1128/MCB.23.16.5706-5715.2003.
Little is known about the quantitative contributions of nonhomologous end joining (NHEJ) and homologous recombination (HR) to DNA double-strand break (DSB) repair in different cell cycle phases after physiologically relevant doses of ionizing radiation. Using immunofluorescence detection of gamma-H2AX nuclear foci as a novel approach for monitoring the repair of DSBs, we show here that NHEJ-defective hamster cells (CHO mutant V3 cells) have strongly reduced repair in all cell cycle phases after 1 Gy of irradiation. In contrast, HR-defective CHO irs1SF cells have a minor repair defect in G(1), greater impairment in S, and a substantial defect in late S/G(2). Furthermore, the radiosensitivity of irs1SF cells is slight in G(1) but dramatically higher in late S/G(2), while V3 cells show high sensitivity throughout the cell cycle. These findings show that NHEJ is important in all cell cycle phases, while HR is particularly important in late S/G(2), where both pathways contribute to repair and radioresistance. In contrast to DSBs produced by ionizing radiation, DSBs produced by the replication inhibitor aphidicolin are repaired entirely by HR. irs1SF, but not V3, cells show hypersensitivity to aphidicolin treatment. These data provide the first evaluation of the cell cycle-specific contributions of NHEJ and HR to the repair of radiation-induced versus replication-associated DSBs.
关于在生理相关剂量的电离辐射后，非同源末端连接（NHEJ）和同源重组（HR）对不同细胞周期阶段DNA双链断裂（DSB）修复的定量贡献，目前所知甚少。我们使用γ-H2AX核灶的免疫荧光检测作为监测DSB修复的新方法，在此表明NHEJ缺陷的仓鼠细胞（CHO突变体V3细胞）在1 Gy照射后的所有细胞周期阶段中修复能力都大幅降低。相比之下，HR缺陷的CHO irs1SF细胞在G(1)期有轻微的修复缺陷，在S期损伤更大，在S期后期/G(2)期有严重缺陷。此外，irs1SF细胞在G(1)期的放射敏感性较低，但在S期后期/G(2)期显著更高，而V3细胞在整个细胞周期中都表现出高敏感性。这些发现表明，NHEJ在所有细胞周期阶段都很重要，而HR在S期后期/G(2)期尤为重要，在该阶段两条途径都对修复和放射抗性有贡献。与电离辐射产生的DSB不同，复制抑制剂阿非迪霉素产生的DSB完全由HR修复。irs1SF细胞而非V3细胞对阿非迪霉素处理表现出超敏感性。这些数据首次评估了NHEJ和HR在细胞周期特异性方面对辐射诱导的与复制相关的DSB修复的贡献。
