Shimura Tsutomu, Martin Melvenia M, Torres Michael J, Gu Cory, Pluth Janice M, DeBernardi Maria A, McDonald Jeffrey S, Aladjem Mirit I
Laboratory of Molecular Pharmacology, Center for Cancer Research, NCI, NIH, Bethesda, MD 20892, USA.
J Mol Biol. 2007 Mar 30;367(3):665-80. doi: 10.1016/j.jmb.2007.01.018. Epub 2007 Jan 12.
Cells that suffer substantial inhibition of DNA replication halt their cell cycle via a checkpoint response mediated by the PI3 kinases ATM and ATR. It is unclear how cells cope with milder replication insults, which are under the threshold for ATM and ATR activation. A third PI3 kinase, DNA-dependent protein kinase (DNA-PK), is also activated following replication inhibition, but the role DNA-PK might play in response to perturbed replication is unclear, since this kinase does not activate the signaling cascades involved in the S-phase checkpoint. Here we report that mild, transient drug-induced perturbation of DNA replication rapidly induced DNA breaks that promptly disappeared in cells that contained a functional DNA-PK whereas such breaks persisted in cells that were deficient in DNA-PK activity. After the initial transient burst of DNA breaks, cells with a functional DNA-PK did not halt replication and continued to synthesize DNA at a slow pace in the presence of replication inhibitors. In contrast, DNA-PK deficient cells subject to low levels of replication inhibition halted cell cycle progression via an ATR-mediated S-phase checkpoint. The ATM kinase was dispensable for the induction of the initial DNA breaks. These observations suggest that DNA-PK is involved in setting a high threshold for the ATR-Chk1-mediated S-phase checkpoint by promptly repairing DNA breaks that appear immediately following inhibition of DNA replication.
遭受DNA复制严重抑制的细胞通过由PI3激酶ATM和ATR介导的检查点反应来停止其细胞周期。目前尚不清楚细胞如何应对较轻的复制损伤，这些损伤处于ATM和ATR激活阈值以下。第三种PI3激酶，即DNA依赖性蛋白激酶（DNA-PK），在复制抑制后也会被激活，但DNA-PK在应对复制紊乱时可能发挥的作用尚不清楚，因为这种激酶不会激活参与S期检查点的信号级联反应。在这里，我们报告说，轻度、短暂的药物诱导的DNA复制扰动会迅速诱导DNA断裂，但在含有功能性DNA-PK的细胞中，这种断裂会迅速消失，而在DNA-PK活性缺陷的细胞中，这种断裂会持续存在。在最初短暂的DNA断裂爆发后，具有功能性DNA-PK的细胞不会停止复制，并且在存在复制抑制剂的情况下继续以缓慢的速度合成DNA。相比之下，受到低水平复制抑制的DNA-PK缺陷细胞通过ATR介导的S期检查点停止细胞周期进程。ATM激酶对于最初的DNA断裂诱导是可有可无的。这些观察结果表明DNA-PK通过迅速修复DNA复制抑制后立即出现的DNA断裂，参与为ATR-Chk1介导的S期检查点设定高阈值。
