Analysis of dynamics of DNA damage repair protein DDB1 in oxidative stresses in N2a cells

我们建立了HEK293细胞系（EGFP-DDB1-HEK293-1），稳定表达egfp融合的DDB1。在EGFP-DDB1-HEK293-1细胞和HEK293细胞中，比较了紫外线诱导的DNA损伤（CPDs和6-4PPs）的修复。因此，它们的修复速度没有差异，说明大量表达DDB1不影响它们的修复。另一方面，两种细胞的细胞分裂速度没有差异。荧光显微镜实时观察氧化应激期EGFP-DDB1-HEK293-1细胞中荧光标记的DDB1从细胞质向细胞核的易位。我们采用添加H_2O_2作为氧化应激。在活细胞中实时测量局部荧光强度，并分析DDB1的动态。另外，还比较了20 J/m^2紫外辐射和1 ~ 20gy电离X射线辐射对DDB1的动力学影响。在X射线电离辐射下，DDB1荧光易位量最大，损伤后24小时荧光易位量达到最大值。损伤48小时后，荧光DDB1转移到细胞质中。这些发现表明，DDB1与多种DNA损伤发生反应，并且DDB1作为一种伴侣蛋白样蛋白将某些信号从细胞质转运到细胞核。另一方面，我们未能从SKN-MC神经母细胞瘤细胞中建立稳定表达egfp融合DDB1的细胞系。提示大量表达全长DDB1可能对SKN-MC神经母细胞瘤细胞有害。由于缺少c端区域的DDB1无法与APP片段结合，APP片段移位到细胞核可能会对神经元细胞造成伤害。

We established a cell line derived from HEK293 (EGFP-DDB1-HEK293-1) stably expressing EGFP-fused DDB1. Both in EGFP-DDB1-HEK293-1 cells and in HEK293 cells, repair of UV-induced DNA damages (CPDs and 6-4PPs) was compared. As a result, there was no difference in the velocity of repair of them, indicating that massive expression of DDB1 did not affect the repair of them. On the other hand, there was no difference in the velocity of cell division in both cells. Translocation of fluorescently labeled DDB1 from cytoplasm to nucleus was on real time observed in the cells of EGFP-DDB1-HEK293-1 after oxidative stresses on the stage kept at 37℃ by means of fluorescence microscopy. As the oxidative stresses, we employed addition of H_2O_2. In living cells, intensity of local fluorescence was measured on real time, and dynamics of DDB1 was analyzed. Otherwise, 20 J/m^2 UV radiation and 1〜20gy ionizing X ray radiation were also compared in dynamics of DDB1. The mount of translocated fluorescent DDB1 was largest in case of ionizing X ray radiation, and the amount of translocated fluorescence was observed in maximum level 24 hours after damages. And fluorescent DDB1 was relocated to cytoplasm 48 hours after damages. These findings indicated that DDB1 reacted with a variety of DNA damages, and that DDB1 functioned as a chaperon-like protein that transported certain signals from cytoplasm to nucleus.On the other hand, we failed to establish a cell line derived from SKN-MC neuroblastoma cells stably expressing EGFP-fused DDB1. It indicated that massive expression of full-length DDB1 might be harmful to SKN-MC neuroblastoma cells. Because DDB1 lack of its C-terminus region has no ability to bind to APP fragments, translocation of APP fragments to nucleus was expected to harm neuronal cells.