Dampening Checkpoint Signaling via Coordinated Action of DNA Repair Scaffolds and the Pph3 Phosphatase

Abstract

During DNA replication, replication forks are prone to stall and collapse. To prevent genomic instability, DNA damage checkpoint (DDC) kinases are activated and coordinate various cellular responses. In S. cerevisiae, the key DDC kinase Rad53 plays crucial roles in the regulation of transcription, dNTP levels, cell cycle progression and origin firing. Importantly, hyperactivation of Rad53 is deleterious and results in sensitivity to replication stress, indicating the importance for regulating Rad53 activity. Recently, we uncovered a phosphatase-independent mechanism for down-regulating Rad53 signaling named DAMP (Dampens Adaptor Mediated-Phosphosignaling). DAMP relies on the Slx4 scaffold protein competing with the checkpoint adaptor Rad9 at sites of lesions to counteract Rad53 activation. Here we show that DAMP functions in parallel with canonical phosphatase mechanisms for Rad53 down-regulation. We show that slx4Δ cells phenocopy cells lacking the main Rad53 phosphatase, Pph3. Both cells show selective sensitivity to methyl methanesulfonate (MMS), accumulate chromosomal defects and hyperactivate Rad53. Both Slx4 and Pph3 seem to converge to the regulation of the Mus81 nuclease, which is necessary for downstream repair. Interestingly, deletion of both SLX4 and PPH3 leads to a synergistic increase in MMS sensitivity and Rad53 activation, suggesting that efficient down-regulation of DDC signaling requires the coordination of DAMP and phosphatases. We propose that these mechanisms operate in distinct spatio-temporal modes, with Slx4 dampening Rad53 activation at lesions, and Pph3 functioning on free, active pools of Rad53 to turn off the checkpoint response.