Shao R G, Cao C X, Zhang H, Kohn K W, Wold M S, Pommier Y
Laboratory of Molecular Pharmacology, Division of Basic Sciences, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892-4255, USA.
EMBO J. 1999 Mar 1;18(5):1397-406. doi: 10.1093/emboj/18.5.1397.
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.
camptothecin/DNA damage/DNA-dependent protein kinase/RPA2 phosphorylation
复制蛋白A（RPA）是一种DNA单链结合蛋白，对DNA复制、重组和修复至关重要。在用拓扑异构酶抑制剂喜树碱或依托泊苷（VP - 16）处理的人类细胞中，我们发现RPA的中等大小亚基RPA2会迅速磷酸化。这种反应似乎是由于DNA依赖性蛋白激酶（DNA - PK）引起的，并且独立于p53或共济失调毛细血管扩张症突变（ATM）蛋白。喜树碱诱导的RPA2磷酸化需要持续的DNA复制。喜树碱本身会部分抑制DNA合成，这种抑制与DNA - PK激活和RPA2磷酸化具有相同的动力学。细胞周期检查点消除剂7 - 羟基星状孢菌素（UCN - 01）可阻止DNA - PK激活和RPA2磷酸化，UCN - 01可显著增强喜树碱的细胞毒性。发现DNA - PK催化亚基（DNA - PKcs）与RPA结合，喜树碱处理后该结合被Ku自身抗原取代。DNA - PKcs在体外直接与RPA1相互作用。我们提出，复制叉与拓扑异构酶 - DNA切割复合物的相遇可能导致复制叉相关的RPA与DNA双链末端相关的DNA - PK并列，从而导致RPA2磷酸化，这可能会向S期检查点机制发出DNA损伤存在的信号。
喜树碱/DNA损伤/DNA依赖性蛋白激酶/RPA2磷酸化
