Phosphorylation of histone H2AX and activation of Mre11, Rad50, and Nbs1 in response to replication-dependent DNA double-strand breaks induced by mammalian DNA topoisomerase I cleavage complexes
Phosphorylation of histone H2AX and activation of Mre11, Rad50, and Nbs1 in response to replication-dependent DNA double-strand breaks induced by mammalian DNA topoisomerase I cleavage complexes
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DOI：
10.1074/jbc.m300198200
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发表时间：
2003-05-30
影响因子：
4.8
通讯作者：
Pommier, Y
中科院分区：
文献类型：
--
作者：
Furuta, T;Takemura, H;Pommier, Y
在真核细胞中，起源于多种原因的DNA双链断裂伴随着磷酸化H2 AX（gammaH 2AX）灶的形成。在这里，我们表明，γ H2 AX的形成也是一个细胞的拓扑异构酶I切割复合物已知的复制过程中诱导DNA双链断裂的反应。在暴露于拓扑异构酶I抑制剂喜树碱的HCT 116人类癌细胞中，所产生的γ H2 AX形成可以用磷脂酰肌醇3-OH激酶相关激酶抑制剂渥曼青霉素来防止;然而，与电离辐射相反，只有喜树碱诱导的γ H2 AX形成可以用DNA复制抑制剂阿非迪霉素来防止，并且用检查点消除剂7-羟基星孢菌素来增强。这种γ H2 AX的形成在ATR（共济失调毛细血管扩张和Rad 3相关）缺陷细胞中受到抑制，在DNA依赖性蛋白激酶缺陷细胞中显著降低，但在共济失调毛细血管扩张细胞中没有消除，表明ATR和DNA依赖性蛋白激酶是在复制介导的DNA双链断裂位点主要参与γ H2 AX形成的激酶。Mre 11和Nbs 1缺陷细胞仍然能够形成γ H2 AX。然而，H2 AX-/-小鼠胚胎成纤维细胞暴露于喜树碱不能形成Mre 11，Rad 50，和Nbs 1灶，是喜树碱过敏。这些结果证明了对于由复制叉碰撞诱导的双链断裂的保守的γ H2 AX应答。γ H2 AX焦点是将修复和检查点蛋白复合物募集到复制断裂位点所必需的。
DNA double-strand breaks originating from diverse causes in eukaryotic cells are accompanied by the formation of phosphorylated H2AX (gammaH2AX) foci. Here we show that gammaH2AX formation is also a cellular response to topoisomerase I cleavage complexes known to induce DNA double-strand breaks during replication. In HCT116 human carcinoma cells exposed to the topoisomerase I inhibitor camptothecin, the resulting gammaH2AX formation can be prevented with the phosphatidylinositol 3-OH kinase-related kinase inhibitor wortmannin; however, in contrast to ionizing radiation, only camptothecin-induced gammaH2AX formation can be prevented with the DNA replication inhibitor aphidicolin and enhanced with the checkpoint abrogator 7-hydroxystaurosporine. This gammaH2AX formation is suppressed in ATR ( ataxia telangiectasia and Rad3-related) deficient cells and markedly decreased in DNA-dependent protein kinase-deficient cells but is not abrogated in ataxia telangiectasia cells, indicating that ATR and DNA-dependent protein kinase are the kinases primarily involved in gammaH2AX formation at the sites of replication-mediated DNA double-strand breaks. Mre11- and Nbs1-deficient cells are still able to form gammaH2AX. However, H2AX-/- mouse embryonic fibroblasts exposed to camptothecin fail to form Mre11, Rad50, and Nbs1 foci and are hypersensitive to camptothecin. These results demonstrate a conserved gammaH2AX response for double-strand breaks induced by replication fork collision. gammaH2AX foci are required for recruiting repair and checkpoint protein complexes to the replication break sites.