Graduate School of Nanobioscience, Yokohama City University, Yokohama, Japan.
PLoS One. 2013 Aug 14;8(8):e72253. doi: 10.1371/journal.pone.0072253. eCollection 2013.
Nonhomologous end-joining (NHEJ) and homologous recombination (HR) are two major pathways for repairing DNA double-strand breaks (DSBs); however, their respective roles in human somatic cells remain to be elucidated. Here we show using a series of human gene-knockout cell lines that NHEJ repairs nearly all of the topoisomerase II- and low-dose radiation-induced DNA damage, while it negatively affects survival of cells harbouring replication-associated DSBs. Intriguingly, we find that loss of DNA ligase IV, a critical NHEJ ligase, and Artemis, an NHEJ factor with endonuclease activity, independently contribute to increased resistance to replication-associated DSBs. We also show that loss of Artemis alleviates hypersensitivity of DNA ligase IV-null cells to low-dose radiation- and topoisomerase II-induced DSBs. Finally, we demonstrate that Artemis-null human cells display increased gene-targeting efficiencies, particularly in the absence of DNA ligase IV. Collectively, these data suggest that DNA ligase IV and Artemis act cooperatively to promote NHEJ, thereby suppressing HR. Our results point to the possibility that HR can only operate on accidental DSBs when NHEJ is missing or abortive, and Artemis may be involved in pathway switching from incomplete NHEJ to HR.
非同源末端连接 (NHEJ) 和同源重组 (HR) 是修复 DNA 双链断裂 (DSBs) 的两种主要途径；然而，它们在人类体细胞中的各自作用仍有待阐明。在这里，我们使用一系列人类基因敲除细胞系表明，NHEJ 修复了拓扑异构酶 II 和低剂量辐射诱导的几乎所有 DNA 损伤，而它对携带复制相关 DSB 的细胞的存活产生负面影响。有趣的是，我们发现关键的 NHEJ 连接酶 DNA 连接酶 IV 和具有内切酶活性的 NHEJ 因子 Artemis 的缺失，独立地导致对复制相关 DSB 的抗性增加。我们还表明，Artemis 的缺失减轻了 DNA 连接酶 IV 缺失细胞对低剂量辐射和拓扑异构酶 II 诱导的 DSB 的超敏反应。最后，我们证明 Artemis 缺失的人类细胞显示出更高的基因靶向效率，尤其是在缺乏 DNA 连接酶 IV 的情况下。总之，这些数据表明 DNA 连接酶 IV 和 Artemis 协同作用以促进 NHEJ，从而抑制 HR。我们的结果表明，当 NHEJ 缺失或无效时，HR 只能作用于偶然的 DSB，并且 Artemis 可能参与了从不完全 NHEJ 到 HR 的途径转换。
