Research Center for Radiation Protection and Fukushima Project Headquarters, National Institute of Radiological Sciences, 4-9-1 Anagawa, Inage-ku, Chiba 263-8555, Japan.
FEBS Open Bio. 2015 Mar 3;5:167-74. doi: 10.1016/j.fob.2015.02.004. eCollection 2015.
In our previous study, a Met-to-Ile substitution at amino acid 256 in the catalytic domain of Uba1 was determined in temperature-sensitive CHO-K1 mutant tsTM3 cells, which exhibited chromosomal instability and cell-cycle arrest in the S to G2 phases with decreased DNA synthesis at the nonpermissive temperature, 39 °C. Mutant cells were also characterized by a significant decrease of Uba1 in the nucleus with decreased ubiquitination activity at 39 °C. Defects of ubiquitination activity in the nucleus resulted in an inappropriate balance between Cdt1 and geminin, a licensing factor of DNA replication and its inhibitor. In the present study, we found that the Cu(I)-catalyzed [3 + 2] cycloaddition (click) reaction inhibits the subsequent indirect immunolabeling of Cdt1 but allows for the detection of PCNA with nascent DNA. Using a procedure without the click reaction, we also demonstrated that Cdt1 remained close to active replication sites in tsTM3 cells at the nonpermissive temperature. Analysis of genome replication by DNA fiber spreading revealed that DNA synthesis continues for at least 10 h after incubation at 39 °C, suggesting that impaired ubiquitination in the nucleus, caused by the defect of Uba1, affected DNA replication only after a long delay.
在我们之前的研究中，确定了 Uba1 催化结构域中氨基酸 256 的 Met 到 Ile 取代，该取代发生在温度敏感型 CHO-K1 突变体 tsTM3 细胞中，该细胞在非允许温度 39°C 时表现出染色体不稳定和 S 期到 G2 期的细胞周期停滞，同时 DNA 合成减少。突变细胞的特点还包括在细胞核中 Uba1 显著减少，同时在 39°C 时泛素化活性降低。核内泛素化活性的缺陷导致 Cdt1 和 geminin 之间的不平衡，Cdt1 是 DNA 复制的许可因子，而 geminin 是其抑制剂。在本研究中，我们发现 Cu(I)催化的[3+2]环加成（点击）反应抑制了随后的 Cdt1 间接免疫标记，但允许用新生 DNA 检测 PCNA。使用没有点击反应的程序，我们还证明 Cdt1 在非允许温度下的 tsTM3 细胞中仍然接近活跃的复制位点。通过 DNA 纤维铺展分析基因组复制，发现 DNA 合成在 39°C 孵育至少 10 小时后仍在继续，这表明 Uba1 的缺陷导致核内泛素化的受损仅在长时间延迟后才会影响 DNA 复制。
