Dampening DNA damage checkpoint signalling via coordinated BRCT domain interactions

In response to DNA damage, checkpoint signalling protects genome integrity at the cost of repressing cell cycle progression and DNA replication. Mechanisms for checkpoint down‐regulation are therefore necessary for proper cellular proliferation. We recently uncovered a phosphatase‐independent mechanism for dampening checkpoint signalling, where the checkpoint adaptor Rad9 is counteracted by the repair scaffolds Slx4‐Rtt107. Here, we establish the molecular requirements for this new mode of checkpoint regulation. We engineered a minimal multi‐BRCT‐domain (MBD) module that recapitulates the action of Slx4‐Rtt107 in checkpoint down‐regulation. MBD mimics the damage‐induced Dpb11‐Slx4‐Rtt107 complex by synergistically interacting with lesion‐specific phospho‐sites in Ddc1 and H2A. We propose that efficient recruitment of Dpb11‐Slx4‐Rtt107 or MBD via a cooperative ‘two‐site‐docking’ mechanism displaces Rad9. MBD also interacts with the Mus81 nuclease following checkpoint dampening, suggesting a spatio‐temporal coordination of checkpoint signalling and DNA repair via a combinatorial mode of BRCT‐domains interactions. Synopsis The DNA repair scaffold proteins Slx4 and Rtt107 utilize a minimal multi‐BRCT‐domain module for phosphatase‐independent downregulation of DNA damage response signals in yeast. Checkpoint dampening by the Rtt107‐Slx4‐Dpb11 complex relies on a ‘two‐site‐docking’ mechanism requiring phosphorylation sites on histone H2A and on the 9‐1‐1 clamp. A minimal BRCT‐domain module (MBD) recapitulates the Rtt107‐Slx4‐Dpb11 complex role in checkpoint dampening and fully rescues MMS sensitivity of cells lacking Slx4. MBD dampens Rad53 activation by specifically counteracting the checkpoint adaptor Rad9. MBD transiently interacts with the 9‐1‐1 clamp and the Mus81 nuclease, but dampens the checkpoint independent of Mus81 function. Graphical Abstract The DNA repair scaffold proteins Slx4 and Rtt107 utilize a minimal multi‐BRCT‐domain module for phosphatase‐independent down‐regulation of DNA damage response signals in yeast.