Olson Erin, Nievera Christian J, Klimovich Vitaly, Fanning Ellen, Wu Xiaohua
Department of Molecular and Experimental Medicine, The Scripps Research Institute, La, Jolla, California 92037, USA.
J Biol Chem. 2006 Dec 22;281(51):39517-33. doi: 10.1074/jbc.M605121200. Epub 2006 Oct 10.
Upon DNA damage, replication is inhibited by the S-phase checkpoint. ATR (ataxia telangiectasia mutated- and Rad3-related) is specifically involved in the inhibition of replicon initiation when cells are treated with DNA damage-inducing agents that stall replication forks, but the mechanism by which it acts to prevent replication is not yet fully understood. We observed that RPA2 is phosphorylated on chromatin in an ATR-dependent manner when replication forks are stalled. Mutation of the ATR-dependent phosphorylation sites in RPA2 leads to a defect in the down-regulation of DNA synthesis following treatment with UV radiation, although ATR activation is not affected. Threonine 21 and serine 33, two residues among several phosphorylation sites in the amino terminus of RPA2, are specifically required for the UV-induced, ATR-mediated inhibition of DNA replication. RPA2 mutant alleles containing phospho-mimetic mutations at ATR-dependent phosphorylation sites have an impaired ability to associate with replication centers, indicating that ATR phosphorylation of RPA2 directly affects the replication function of RPA. Our studies suggest that in response to UV-induced DNA damage, ATR rapidly phosphorylates RPA2, disrupting its association with replication centers in the S-phase and contributing to the inhibition of DNA replication.
在DNA损伤时，S期检查点会抑制复制。当细胞用导致复制叉停滞的DNA损伤诱导剂处理时，共济失调毛细血管扩张症突变和Rad3相关蛋白（ATR）特别参与复制子起始的抑制，但它阻止复制的作用机制尚未完全清楚。我们观察到，当复制叉停滞时，RPA2在染色质上以ATR依赖的方式被磷酸化。RPA2中ATR依赖的磷酸化位点发生突变，尽管ATR激活不受影响，但在用紫外线辐射处理后，DNA合成的下调出现缺陷。RPA2氨基末端几个磷酸化位点中的苏氨酸21和丝氨酸33，是紫外线诱导的、ATR介导的DNA复制抑制所特别需要的。在ATR依赖的磷酸化位点含有模拟磷酸化突变的RPA2突变等位基因与复制中心结合的能力受损，这表明RPA2的ATR磷酸化直接影响RPA的复制功能。我们的研究表明，响应紫外线诱导的DNA损伤时，ATR迅速磷酸化RPA2，破坏其在S期与复制中心的结合，并导致DNA复制受到抑制。
