Termination of replication stress signaling via concerted action of the Slx4-Rtt107 scaffolds and the PP4 phosphatase. Saccharomyces cerevisiae

In response to replication stress, signaling mediated by DNA damage checkpoint kinases protects genome integrity. However, following repair or bypass of DNA lesions, checkpoint signaling needs to be terminated for continued cell cycle progression and proliferation. In budding yeast, the PP4 phosphatase has been shown to play a key role in preventing hyperactivation of the checkpoint kinase Rad53. In addition, we recently uncovered a phosphatase-independent mechanism for down-regulating Rad53 in which the DNA repair scaffolds Slx4-Rtt107 decrease engagement of the checkpoint adaptor Rad9 at DNA lesions. Here we reveal that proper termination of checkpoint signaling following the bypass of replication blocks imposed by alkylated DNA adducts requires the concerted action of these two fundamentally distinct mechanisms of checkpoint down-regulation. Cells lacking both SLX4 and the PP4-subunit Pph3 display a synergistic increase in Rad53 signaling and are exquisitely sensitive to the DNA alkylating agent methyl methanesulfonate, which induces replication blocks and extensive formation of chromosomal linkages due to template switching mechanisms required for fork bypass. Rad53 hypersignaling in these cells seems to converge to a strong repression of Mus81-Mms4, the nuclease responsible for resolving chromosomal linkages, thus explaining the selective sensitivity of slx4Δpph3Δ cells to alkylation damage. Our results support a model in which Slx4-Rtt107 act locally to down-regulate Rad53 activation following fork bypass, while PP4 acts on pools of active Rad53 (and histone H2A phosphorylated on Ser129) that have diffused from the site of lesion. We propose that the proper spatial coordination of Slx4-Rtt107 and PP4 action is crucial to allow timely activation of Mus81-Mms4 and, therefore, proper chromosome segregation.