Department of Life Sciences, Chung-Ang University, Seoul, 156-756, Korea.
Department of Biomedical Science, CHA University, Seoul, Korea.
Sci Rep. 2017 Sep 14;7(1):11610. doi: 10.1038/s41598-017-11951-1.
Embryonic stem (ES) cells require homologous recombination (HR) to cope with genomic instability caused during self-renewal. Here, we report expression dynamics and localization of endogenous HR factors in DNA break repair of ES cells. In addition, we analyzed gene expression patterns of HR-related factors at the transcript level with RNA-sequencing experiments. We showed that ES cells constitutively expressed diverse HR proteins throughout the cell cycle and that HR protein expression was not significantly changed even in the DNA damaging conditions. We further analyzed that depleting Rad51 resulted in the accumulation of larger single-stranded DNA (ssDNA) gaps, but did not perturb DNA replication, indicating that ES cells were able to enter the G2-phase in the presence of unrepaired DNA gaps, consistent with the possibility that post-replication repair helps avoid stalling at the G2/M checkpoint. Interestingly, caffeine treatment inhibited the formation of Rad51 or Rad54 foci, but not the formation of γH2AX and Exo1 foci, which led to incomplete HR in ssDNA, thus increasing DNA damage sensitivity. Our results suggested that ES cells possess conserved HR-promoting machinery to ensure effective recruitment of the HR proteins to DNA breaks, thereby driving proper chromosome duplication and cell cycle progression in ES cells.
胚胎干细胞 (ES) 细胞需要同源重组 (HR) 来应对自我更新过程中产生的基因组不稳定性。在这里，我们报告了内源性 HR 因子在 ES 细胞 DNA 断裂修复中的表达动态和定位。此外，我们还通过 RNA-seq 实验分析了 HR 相关因子在转录水平上的基因表达模式。结果表明，ES 细胞在整个细胞周期中持续表达多种 HR 蛋白，即使在 DNA 损伤条件下，HR 蛋白的表达也没有明显变化。我们进一步分析表明，Rad51 的耗竭导致更大的单链 DNA (ssDNA) 缺口积累，但不会干扰 DNA 复制，这表明 ES 细胞能够在未修复的 DNA 缺口存在的情况下进入 G2 期，这与复制后修复有助于避免在 G2/M 检查点停滞的可能性一致。有趣的是，咖啡因处理抑制了 Rad51 或 Rad54 焦点的形成，但不抑制 γH2AX 和 Exo1 焦点的形成，这导致 ssDNA 中的 HR 不完全，从而增加了 DNA 损伤敏感性。我们的结果表明，ES 细胞具有保守的 HR 促进机制，以确保 HR 蛋白有效地招募到 DNA 断裂处，从而驱动 ES 细胞中正确的染色体复制和细胞周期进程。
