A combined computational and biochemical approach reveals how mitotic kinases allow cell division to proceed in the presence of DNA damage.
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 is constantly damaged both by factors outside our bodies (such as ultraviolet rays from sunlight) and by factors from within (such as reactive oxygen species produced during metabolism). DNA damage can lead to malfunctioning of genes, and persistent DNA damage can result in developmental disorders or the development of cancer. To ensure proper DNA repair, cells are equipped with an evolutionarily conserved DNA damage checkpoint, which stops proliferation and activates DNA repair mechanisms. Intriguingly, this DNA damage checkpoint responds to DNA damage throughout the cell cycle, except during mitosis. In this work, we have addressed how cells dismantle their DNA damage checkpoint during mitosis to allow cell division to proceed even if there is damaged DNA present. Using the observation that kinases phosphorylate their substrates on evolutionarily conserved, kinase-specific sequence motifs, we have used a combined computational and experimental approach to predict and verify key proteins involved in mitotic checkpoint inactivation. We show that the checkpoint scaffold protein 53BP1 is phosphorylated by the mitotic kinases Cdk1 and Polo-like kinase-1 (Plk1). Furthermore, we find that Plk1 can inactivate the checkpoint kinase Chk2, which is downstream of 53BP1. Plk1 is shown to be a key mediator of mitotic checkpoint inactivation, as cells that cannot activate Plk1 fail to properly dismantle the DNA damage checkpoint during mitosis and instead show DNA damage-induced Chk2 kinase activation. Two related papers, published in PLoS Biology (Vidanes et al., doi:10.1371/journal.pbio.1000286) and PLoS Genetics (Donnianni et al., doi:10.1371/journal.pgen.1000763), similarly investigate the phenomenon of DNA damage checkpoint silencing.