Replication protein A2 phosphorylation after DNA damage by the coordinated action of ataxia telangiectasia-mutated and DNA-dependent protein kinase.
Replication protein A2 phosphorylation after DNA damage by the coordinated action of ataxia telangiectasia-mutated and DNA-dependent protein kinase.
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DOI：
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发表时间：
2001-12
期刊：
影响因子：
11.2
通讯作者：
H. Wang;J. Guan;H. Wang;A. R. Perrault;Y. Wang;G. Iliakis
中科院分区：
文献类型：
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作者：
H. Wang;J. Guan;H. Wang;A. R. Perrault;Y. Wang;G. Iliakis
复制蛋白A（RPA，也称为人单链DNA结合蛋白）是一种三聚体，多功能蛋白质复合物，参与DNA复制，DNA修复和重组。在细胞暴露于电离辐射（IR）和其他DNA损伤剂后观察到RPA 2亚基的磷酸化，这暗示修饰的蛋白质在DNA损伤后或在DNA修复中调节DNA复制。虽然共济失调毛细血管扩张症突变（ATM）和DNA依赖性蛋白激酶（DNA-PK）磷酸化RPA 2在体外，他们在体内的作用仍然不确定，矛盾的结果已经报道。在这里，我们表明，RPA 2磷酸化是延迟在这些激酶之一的缺陷细胞和野生型，ATM，或DNA-PK缺陷细胞治疗后，在抑制ATM和DNA-PK的浓度渥曼青霉素完全废除。咖啡因是ATM和ATM-Rad 3相关（ATR）的抑制剂，但不是DNA-PK的抑制剂，在野生型细胞中产生共济失调-毛细血管扩张样反应，完全阻止DNA-PKcs缺陷细胞中RPA 2的磷酸化，但对共济失调-毛细血管扩张细胞没有影响。这些观察结果排除了ATR，并暗示ATM和DNA-PK在暴露于IR后的RPA 2磷酸化中。UCN-01是蛋白激酶C、Chk 1和细胞周期蛋白依赖性激酶的抑制剂，对IR诱导的RPA 2磷酸化没有影响。由于UCN-01消除了检查点应答，因此该观察结果将RPA 2磷酸化与检查点激活分离。磷酸化RPA对核结构的亲和力高于未磷酸化RPA，表明蛋白质的功能改变。在DNA复制的体外试验中，DNA-PK是磷酸化RPA 2的唯一激酶，表明ATM磷酸化RPA 2需要体外试验中未重现的过程。由于RPA 2的磷酸化动力学与S期检查点的磷酸化动力学不同，我们提出DNA-PK和ATM在DNA损伤后协同磷酸化RPA，将蛋白质的功能从DNA复制重定向到DNA修复。
Replication protein A (RPA, also known as human single-stranded DNA-binding protein) is a trimeric, multifunctional protein complex involved in DNA replication, DNA repair, and recombination. Phosphorylation of the RPA2 subunit is observed after exposure of cells to ionizing radiation (IR) and other DNA-damaging agents, which implicates the modified protein in the regulation of DNA replication after DNA damage or in DNA repair. Although ataxia telangiectasia-mutated (ATM) and DNA-dependent protein kinase (DNA-PK) phosphorylate RPA2 in vitro, their role in vivo remains uncertain, and contradictory results have been reported. Here we show that RPA2 phosphorylation is delayed in cells deficient in one of these kinases and completely abolished in wild-type, ATM, or DNA-PK-deficient cells after treatment with wortmannin at a concentration-inhibiting ATM and DNA-PK. Caffeine, an inhibitor of ATM and ATM-Rad3 related (ATR) but not DNA-PK, generates an ataxia-telangiectasia-like response in wild-type cells, prevents completely RPA2 phosphorylation in DNA-PKcs deficient cells, but has no effect on ataxia-telangiectasia cells. These observations rule out ATR and implicate both ATM and DNA-PK in RPA2 phosphorylation after exposure to IR. UCN-01, an inhibitor of protein kinase C, Chk1, and cyclin-dependent kinases, has no effect on IR-induced RPA2 phosphorylation. Because UCN-01 abrogates checkpoint responses, this observation dissociates RPA2 phosphorylation from checkpoint activation. Phosphorylated RPA has a higher affinity for nuclear structures than unphosphorylated RPA suggesting functional alterations in the protein. In an in vitro assay for DNA replication, DNA-PK is the sole kinase phosphorylating RPA2, indicating that processes not reproduced in the in vitro assay are required for RPA2 phosphorylation by ATM. Because RPA2 phosphorylation kinetics are distinct from those of the S phase checkpoint, we propose that DNA-PK and ATM cooperate to phosphorylate RPA after DNA damage to redirect the functions of the protein from DNA replication to DNA repair.