Replication-mediated DNA damage by camptothecin induces phosphorylation of RPA by DNA-dependent protein kinase and dissociates RPA:DNA-PK complexes
Replication-mediated DNA damage by camptothecin induces phosphorylation of RPA by DNA-dependent protein kinase and dissociates RPA:DNA-PK complexes
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DOI：
10.1093/emboj/18.5.1397
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发表时间：
1999-03-01
期刊：
影响因子：
11.4
通讯作者：
Pommier, Y
中科院分区：
文献类型：
--
作者：
Shao, RG;Cao, CX;Pommier, Y
复制蛋白A（RPA）是DNA复制、重组和修复所必需的DNA单链结合蛋白。在用拓扑异构酶抑制剂喜树碱或依托泊苷（VP-16）处理的人细胞中，我们发现RPA的中等大小的亚基RPA 2迅速磷酸化，这种反应似乎是由于DNA依赖性蛋白激酶（DNA-PK），而不依赖于p53或共济失调毛细血管扩张突变（ATM）蛋白。响应喜树碱的RPA 2磷酸化需要持续的DNA复制。喜树碱本身部分抑制DNA合成，并且这种抑制遵循与DNA-PK活化和RPA 2磷酸化相同的动力学，DNA-PK活化和RPA 2磷酸化被细胞周期检查点消除剂7-羟基星形孢菌素（UCN-01）阻止，其显著增强喜树碱的细胞毒性。发现DNA-PK催化亚基（DNA-PKcs）结合RPA，其在喜树碱处理后被Ku自身抗原取代。DNA-PKcs在体外与RPA 1直接相互作用。我们提出，遇到的复制叉与拓扑异构酶-DNA切割复合物可能会导致并列的复制叉相关RPA和DNA双链末端相关的DNA-PK，导致RPA 2磷酸化，这可能是信号的存在下，DNA损伤的S期检查点机制。
Replication protein A (RPA) is a DNA single-strand binding protein essential for DNA replication, recombination and repair. In human cells treated with the topoisomerase inhibitors camptothecin or etoposide (VP-16), we find that RPA2, the middle-sized subunit of RPA, becomes rapidly phosphorylated, This response appears to be due to DNA-dependent protein kinase (DNA-PK) and to be independent of p53 or the ataxia telangiectasia mutated (ATM) protein. RPA2 phosphorylation in response to camptothecin required ongoing DNA replication. Camptothecin itself partially inhibited DNA synthesis, and this inhibition followed the same kinetics as DNA-PK activation and RPA2 phosphorylation, DNA-PK activation and RPA2 phosphorylation were prevented by the cell-cycle checkpoint abrogator 7-hydroxystaurosporine (UCN-01), which markedly potentiates camptothecin cytotoxicity. The DNA-PK catalytic subunit (DNA-PKcs) was found to bind RPA which was replaced by the Ku autoantigen upon camptothecin treatment. DNA-PKcs interacted directly with RPA1 in vitro. We propose that the encounter of a replication fork with a topoisomerase-DNA cleavage complex could lead to a juxtaposition of replication fork-associated RPA and DNA double-strand end-associated DNA-PK, leading to RPA2 phosphorylation which may signal the presence of DNA damage to an S-phase checkpoint mechanism.