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DNA damage elicits a checkpoint response depending on the Mec1/ATR kinase, which detects the presence of single-stranded DNA and activates the effector kinase Rad53/CHK2. In Saccharomyces cerevisiae, one of the signaling circuits leading to Rad53 activation involves the evolutionarily conserved 9-1-1 complex, which acts as a platform for the binding of Dpb11 and Rad9 (referred to as the 9-1-1 axis) to generate a protein complex that allows Mec1 activation. By examining the effects of both loss-of-function and hypermorphic mutations, here, we show that the Cdc55 and Tpd3 subunits of the PP2A phosphatase counteract activation of the 9-1-1 axis. The lack of this inhibitory function results in DNA-damage sensitivity, sustained checkpoint-mediated cell-cycle arrest, and impaired resection of DNA double-strand breaks. This PP2A anti-checkpoint role depends on the capacity of Cdc55 to interact with Ddc1 and to counteract Ddc1-Dpb11 complex formation by preventing Dpb11 recognition of Ddc1 phosphorylated on Thr602.

Homologous recombination is initiated by nucleolytic degradation (resection) of DNA double-strand breaks (DSBs). DSB resection is a two-step process in which an initial short-range step is catalyzed by the Mre11-Rad50-Xrs2 (MRX) complex and limited to the vicinity of the DSB end. Then the two long-range resection Exo1 and Dna2-Sgs1 nucleases extend the resected DNA tracts. How short-range resection is regulated and contributes to checkpoint activation remains to be determined. Here, we show that abrogation of long-range resection induces a checkpoint response that decreases DNA damage resistance. This checkpoint depends on the 9-1-1 complex, which recruits Dpb11 and Rad9 at damaged DNA. Furthermore, the 9-1-1 complex, independently of Dpb11 and Rad9, restricts short-range resection by negatively regulating Mre11 nuclease. We propose that 9-1-1, which is loaded at the leading edge of resection, plays a key function in regulating Mre11 nuclease and checkpoint activation once DSB resection is initiated.