David H. Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA.
PLoS Biol. 2010 Jan 26;8(1):e1000287. doi: 10.1371/journal.pbio.1000287.
DNA damage checkpoints arrest cell cycle progression to facilitate DNA repair. The ability to survive genotoxic insults depends not only on the initiation of cell cycle checkpoints but also on checkpoint maintenance. While activation of DNA damage checkpoints has been studied extensively, molecular mechanisms involved in sustaining and ultimately inactivating cell cycle checkpoints are largely unknown. Here, we explored feedback mechanisms that control the maintenance and termination of checkpoint function by computationally identifying an evolutionary conserved mitotic phosphorylation network within the DNA damage response. We demonstrate that the non-enzymatic checkpoint adaptor protein 53BP1 is an in vivo target of the cell cycle kinases Cyclin-dependent kinase-1 and Polo-like kinase-1 (Plk1). We show that Plk1 binds 53BP1 during mitosis and that this interaction is required for proper inactivation of the DNA damage checkpoint. 53BP1 mutants that are unable to bind Plk1 fail to restart the cell cycle after ionizing radiation-mediated cell cycle arrest. Importantly, we show that Plk1 also phosphorylates the 53BP1-binding checkpoint kinase Chk2 to inactivate its FHA domain and inhibit its kinase activity in mammalian cells. Thus, a mitotic kinase-mediated negative feedback loop regulates the ATM-Chk2 branch of the DNA damage signaling network by phosphorylating conserved sites in 53BP1 and Chk2 to inactivate checkpoint signaling and control checkpoint duration.
DNA 损伤检查点会阻止细胞周期进程，以促进 DNA 修复。能否在遗传毒性损伤后存活不仅取决于细胞周期检查点的启动，还取决于检查点的维持。虽然 DNA 损伤检查点的激活已被广泛研究，但维持和最终失活细胞周期检查点的分子机制在很大程度上仍是未知的。在这里，我们通过计算识别 DNA 损伤反应中的一个进化上保守的有丝分裂磷酸化网络，探索了控制检查点功能维持和终止的反馈机制。我们证明非酶促检查点衔接蛋白 53BP1 是细胞周期激酶细胞周期蛋白依赖性激酶 1 和 Polo 样激酶 1 (Plk1) 的体内靶标。我们表明，Plk1 在有丝分裂期间与 53BP1 结合，这种相互作用对于正确失活 DNA 损伤检查点是必需的。无法结合 Plk1 的 53BP1 突变体在电离辐射介导的细胞周期阻滞后无法重新启动细胞周期。重要的是，我们表明 Plk1 还磷酸化 53BP1 结合的检查点激酶 Chk2，以失活其 FHA 结构域并抑制其在哺乳动物细胞中的激酶活性。因此，有丝分裂激酶介导的负反馈环通过磷酸化 53BP1 和 Chk2 中的保守位点来失活检查点信号，并控制检查点持续时间，从而调节 DNA 损伤信号网络中的 ATM-Chk2 分支。
