Department of Biological Sciences, Ibaraki University, Bunkyo 2-1-1, Mito, Ibaraki 310-8512, Japan.
Department of Cancer Biology, Nagoya University Graduate School of Medicine, 65 Tsurumai-cho, Showa-ku, Nagoya 466-8550, Japan.
J Radiat Res. 2021 Jan 1;62(1):25-33. doi: 10.1093/jrr/rraa095.
The choice of repair pathways of DNA double-strand breaks (DSBs) is dependent upon the cell cycle phases. While homologous recombination repair (HRR) is active between the S and G2 phases, its involvement in mitotic DSB repair has not been examined in detail. In the present study, we developed a new reporter assay system to detect homology-directed repair (HDR), a major pathway used for HRR, in combination with an inducible DSB-generation system. As expected, the maximal HDR activity was observed in the late S phase, along with minimal activity in the G1 phase and at the G1/S boundary. Surprisingly, significant HDR activity was observed in M phase, and the repair efficiency was similar to that observed in late S phase. HDR was also confirmed in metaphase cells collected with continuous colcemid exposure. ChIP assays revealed the recruitment of RAD51 to the vicinity of DSBs in M phase. In addition, the ChIP assay for gamma-H2AX and phosphorylated DNA-PKcs indicated that a part of M-phase cells with DSBs could proceed into the next G1 phase. These results provide evidence showing that a portion of mitotic cell DSBs are undoubtedly repaired through action of the HDR repair pathway.
DNA 双链断裂 (DSB) 的修复途径选择取决于细胞周期阶段。虽然同源重组修复 (HRR) 在 S 和 G2 期之间活跃，但它在有丝分裂 DSB 修复中的作用尚未详细研究。在本研究中，我们开发了一种新的报告基因检测系统，用于检测同源定向修复 (HDR)，这是 HRR 的主要途径，与诱导性 DSB 生成系统相结合。正如预期的那样，最大的 HDR 活性出现在晚期 S 期，而在 G1 期和 G1/S 边界处活性最低。令人惊讶的是，在 M 期观察到显著的 HDR 活性，其修复效率与晚期 S 期相似。在连续秋水仙素暴露收集的中期细胞中也证实了 HDR。ChIP 分析显示 RAD51 在 M 期被募集到 DSB 附近。此外，γ-H2AX 和磷酸化 DNA-PKcs 的 ChIP 分析表明，一部分带有 DSB 的 M 期细胞可以进入下一个 G1 期。这些结果提供了证据，表明有丝分裂细胞 DSB 的一部分无疑是通过 HDR 修复途径修复的。
