Borda Miguel Angel, Palmitelli Micaela, Verón Gustavo, González-Cid Marcela, de Campos Nebel Marcelo
Laboratorio de Mutagénesis, Instituto de Medicina Experimental (CONICET-Academia Nacional de Medicina), Buenos Aires, Argentina.
Laboratorio de Mutagénesis, Instituto de Medicina Experimental (CONICET-Academia Nacional de Medicina), Buenos Aires, Argentina.
Mutat Res. 2015 Nov;781:37-48. doi: 10.1016/j.mrfmmm.2015.09.003. Epub 2015 Sep 14.
Tyrosyl-DNA-phosphodiesterase 1 (TDP1) is a DNA repair enzyme that removes irreversible protein-linked 3' DNA complexes, 3' phosphoglycolates, alkylation damage-induced DNA breaks, and 3' deoxyribose nucleosides. In addition to its extended spectrum of substrates, TDP1 interacts with several DNA damage response factors. To determine whether TDP1 participates in the repair of topoisomerase II (Top2) induced DNA lesions, we generated TDP1 depleted (TDP1kd) human tumoral cells. We found that TDP1kd cells are hypersensitive to etoposide (ETO). Moreover, we established in a chromatin context that following treatment with ETO, TDP1kd cells accumulate increased amounts of Top2α cleavage complexes, removing them with an altered kinetics. We also showed that TDP1 depleted cells accumulate increased γH2AX and pS296Chk1 signals following treatment with ETO. Similarly, cytogenetics analyses following Top2 poisoning revealed increased amounts of chromatid and chromosome breaks and exchanges on TDP1kd cells in the presence or not of the DNA-PKcs inhibitor NU7026. However, the levels of sister chromatid exchanges were similar in both TDP1kd and control non-silenced cell lines. This suggests a role of TDP1 in both canonical non-homologous end joining and alternative end joining, but not in the homologous recombination repair pathway. Finally, micronucleus analyses following ETO treatment revealed a higher frequency of micronucleus containing γH2AX signals on TDP1kd cells. Together, our results highlight an active role of TDP1 in the repair of Top2-induced DNA damage and its relevance on the genome stability maintenance in human cells.
酪氨酰 - DNA - 磷酸二酯酶1(TDP1)是一种DNA修复酶,可去除不可逆的蛋白质连接的3' DNA复合物、3' 磷酸乙醇酸、烷基化损伤诱导的DNA断裂以及3' 脱氧核糖核苷。除了其广泛的底物谱外,TDP1还与多种DNA损伤反应因子相互作用。为了确定TDP1是否参与拓扑异构酶II(Top2)诱导的DNA损伤修复,我们构建了TDP1缺失(TDP1kd)的人肿瘤细胞。我们发现TDP1kd细胞对依托泊苷(ETO)高度敏感。此外,我们在染色质环境中证实,用ETO处理后,TDP1kd细胞中Top2α切割复合物的积累量增加,且清除动力学发生改变。我们还表明,用ETO处理后,TDP1缺失的细胞中γH2AX和pS296Chk1信号积累增加。同样,Top2中毒后的细胞遗传学分析显示,无论是否存在DNA - PKcs抑制剂NU7026,TDP1kd细胞中的染色单体和染色体断裂及交换量均增加。然而,TDP1kd细胞系和对照非沉默细胞系中的姐妹染色单体交换水平相似。这表明TDP1在经典的非同源末端连接和替代末端连接中均起作用,但在同源重组修复途径中不起作用。最后,ETO处理后的微核分析显示,TDP1kd细胞中含有γH2AX信号的微核频率更高。总之,我们的结果突出了TDP1在Top2诱导的DNA损伤修复中的积极作用及其与人类细胞基因组稳定性维持的相关性。