Fen1 is induced p53 dependently and involved in the recovery from UV- light-induced replication inhibition
Fen1 is induced p53 dependently and involved in the recovery from UV- light-induced replication inhibition
复制标题
DOI：
10.1038/sj.onc.1208994
复制
发表时间：
2005-12-15
期刊：
影响因子：
8
通讯作者：
Kaina, B
中科院分区：
文献类型：
--
作者：
Christmann, M;Tomicic, MT;Kaina, B
缺乏 p53 的小鼠胚胎成纤维细胞 (MEF) 对紫外线 (UV-C) 的细胞毒性和基因毒性作用高度敏感。它们在从 UV-C 诱导的 DNA 复制抑制中恢复方面也表现出缺陷。参与处理停滞 DNA 复制叉的酶是瓣核酸内切酶 1 (Fen1)。基因表达亲。 UV-C 照射的 MEF 的处理显示 fen1 上调，RT-PCR 和 Western blot 实验证实了这一点。抑制剂研究表明，UV-C 暴露后 Fen1 水平升高是由于转录激活所致。 Fen1 诱导具有剂量和时间依赖性；辐射后 3 小时就在蛋白质水平上发生了。 UV-C 诱导 Fen1 需要 p53，因为它在 p53 野生型 (wt) 中观察到，但在 p53 缺失 (p53-/-) 成纤维细胞中没有观察到。在复制型野生细胞中，Fen1 的上调与 p53 蛋白水平的增加平行，而在非复制型细胞中，Fen1 和 p53 均不受 UV-C 诱导。小鼠 fen1 启动子被克隆并显示含有 p53 结合的 p53 共有序列。在共转染实验中，p53 刺激 fen1 启动子-报告基因构建体的表达。 P53 缺失细胞中 Fen1 的转基因表达减弱了 UV-C 光诱导的 DNA 复制抑制，支持了 Fen1 诱导参与细胞从 DNA 损伤中恢复的假设。
Mouse embryonic fibroblasts (MEFs) that lack p53 are hypersensitive to the cytotoxic and genotoxic effect of ultraviolet (UV-C) light. They also display a defect in the recovery from UV-C-induced DNA replication inhibition. An enzyme involved in processing stalled DNA replication forks is flap endonuclease 1 (Fen1). Gene expression pro. ling of UV-C-irradiated MEFs revealed fen1 to be upregulated, which was confirmed by RT-PCR and Western blot experiments. Increased Fen1 levels upon UV-C exposure are due to transcriptional activation, as revealed by inhibitor studies. Fen1 induction was dose- and time-dependent; it occurred on protein level already 3 h after irradiation. Induction of Fen1 by UV-C requires p53 since it was observed in p53 wild-type (wt) but not in p53 null (p53-/-) fibroblasts. Fen1 upregulation paralleled the increase in p53 protein level in replicating wt cells, whereas in nonreplicating cells both Fen1 and p53 were not induced by UV-C. The mouse fen1 promoter was cloned and shown to harbor a p53 consensus sequence to which p53 binds. In cotransfection experiments, p53 stimulated the expression of a fen1 promoter-reporter construct. Transgenic expression of Fen1 in p53 null cells attenuated UV-C light-induced DNA replication inhibition, supporting the hypothesis that Fen1 induction is involved in the recovery of cells from DNA damage.