Inactivation of Rad53 allows budding yeast cells to recover from checkpoint-mediated cell cycle arrest. To date, less is known about the mechanisms that silence the checkpoint after inhibition of DNA replication by nucleotide depletion. Here, our results indicate that Glc7/PP1 promotes recovery from inhibition of DNA replication, and this control is linked to γH2A downregulation and Rad53 inactivation. In fact, defective Glc7 impairs resumption of DNA replication after HU-induced fork stalling, and this defect correlates with persistent Rad53 hyperphosphorylation. The latter does not seem to be due to defects in repairing DSBs that may arise as a consequence of replication fork stalling, as glc7
mutants are proficient in DSB repair by HR. Moreover, the artificial inactivation of Rad53 suppresses the replication recovery defects of glc7-T152K
mutant cells, indicating that the critical function of Glc7 in replication resumption is likely Rad53 dephosphorylation. This finding also implies that the restart of DNA synthesis at stalled replication forks is coupled to Rad53 deactivation. Thus, although Rad53 activation plays a crucial role in the stabilization of stalled replication forks, possibly by phosphorylating DNA replication proteins (reviewed in reference 42
), its subsequent Glc7-mediated inactivation is important for replication fork restart after HU treatment. Similarly, Rad53 deactivation was shown to allow replication fork restart in pph3
Δ cells recovering from MMS-induced DNA damage (32
Interestingly, although MMS treatment interferes with S phase progression similarly to nucleotide depletion, Glc7 does not seem to promote Rad53 dephosphorylation after MMS exposure. On the contrary, the phosphatases Ptc2, Ptc3, and Pph3, which are required to turn off the checkpoint induced by MMS, are dispensable for replication recovery during HU-induced S phase arrest (26
). Furthermore, the lack of Pph3 or of both Ptc2 and Ptc3 did not enhance either the sensitivity to HU of glc7-T152K
cells or their defects in completing DNA replication when exposed to low HU dose. These findings suggest that different phosphatases might be specifically engaged in response to particular types of signals. Interestingly, cells concomitantly lacking Pph3, Ptc2, and Ptc3 show S phase progression defects and hyperphosphorylated Rad53 accumulation even in the absence of genotoxic treatment, and their growth defects are greatly enhanced by the presence of the glc7-T152K
allele. Thus, Glc7, Pph3, Ptc2, and Ptc3 appear to act redundantly in supporting cell viability under unperturbed conditions.
Glc7 function in checkpoint termination is not limited to the checkpoint triggered by nucleotide depletion. In fact, Glc7 overproduction causes premature disappearance of phosphorylated Rad53 after phleomycin-induced DSB formation. Furthermore, the lack of Glc7 impairs resumption of cell cycle progression and Rad53 dephosphorylation under the same conditions, indicating that it is involved also in recovery from a DSB-induced checkpoint. Interestingly, Glc7 seems to act redundantly with Pph3 in checkpoint deactivation in the response to DSBs, as the glc7-T152K allele enhances the sensitivity to phleomycin of pph3Δ cells, but not their sensitivity to HU (Fig. ).
How does Glc7 promote termination of the checkpoint response? A checkpoint signal is triggered by the recruitment to stalled replication forks or DSB sites of the Mec1 and Tel1 checkpoint kinases, which phosphorylate H2A, Ddc2, Rad9, and Mre11 proteins, thus propagating the checkpoint signal to the downstream kinase Rad53. Rad53 phosphorylation and activation require Ddc2, Rad9, and Mre11, while γH2A, Rad9, Ddc2, and Mre11 phosphorylation occurs independently of Rad53 (9
). We find that Glc7 counteracts phosphorylation not only of Rad53 but also of γH2A, Ddc2, Rad9, and Mre11 after HU exposure or chemically induced DSBs, suggesting that Glc7 can reverse phosphorylation of Rad53 and/or of proteins upstream. However, since there is no evidence that Rad53 regulates γH2A, Ddc2, Mre11, and Rad9 phosphorylation in a positive feedback loop, it seems unlikely that Glc7 promotes checkpoint termination by acting exclusively on Rad53 phosphosites. On the other hand, Glc7 dephosphorylates γH2A in vitro, and the lack of γH2A formation not only counteracts Rad53 phosphorylation in HU-treated glc7-T152K
cells but also partially suppresses their defects in replication recovery. Therefore, we favor the hypothesis that Glc7 may promote resumption of DNA replication by reversing γH2A formation. However, we cannot exclude the possibility that Glc7 is involved in checkpoint termination indirectly, by regulating multiple targets in the DNA damage response. Indeed, Glc7 is known to target a wide range of substrates through interactions with specific factors, and the analyzed glc7
mutations, which map on the protein surface (3
), might affect protein-protein interactions. Identification of the factor(s) possibly targeting Glc7 to γH2A will shed light on the mechanistic connections between histone modifications, DNA replication, and checkpoint signaling.
How can γH2A dephosphorylation promote resumption of DNA replication? Formation of γH2A is dispensable for checkpoint activation when the dNTP pool is limiting. However, the lack of γH2A in HU-treated glc7-T152K cells results in reduced Rad53 phosphorylation, indicating that the signal keeping Rad53 active in glc7 cells consists in γH2A molecules. Thus, γH2A formation supports the maintenance of Rad53 activation, possibly by promoting congregation of DNA damage response proteins at stalled replication forks. As artificial Rad53 inactivation suppresses the defects of glc7-T152K cells in replication recovery, removal of γH2A may reduce the checkpoint signal to a level below which it is unable to activate Rad53, thus permitting replication fork restart.
As the lack of γH2A has been shown to contribute to recovery from a DSB-induced checkpoint (17
), γH2A persistence in phleomycin-treated glc7
mutants might account also for their defects in recovering from DSBs. However, the lack of γH2A formation does not suppress the hypersensitivity to phleomycin of the glc7-T152K
mutant, suggesting that their increased sensitivity may be due to Glc7 functions in the DNA damage response other than γH2A dephosphorylation. Consistent with this hypothesis, it has been shown that Schizosaccharomyces pombe
PP1/Dis2 dephosphorylates the checkpoint kinase Chk1 (10
) and mammalian PP1a has been shown to dephosphorylate a fragment of BRCA1 in vitro (19
In summary, we have found that the yeast PP1 phosphatase Glc7 is involved in checkpoint termination after both HU-induced replication block and DSBs and that the critical function of Glc7 in this process is dephosphorylation of γH2A. The emerging view is that different sets of phosphatases participate in checkpoint inactivation after specific types of replication stress. Unraveling how these phosphatases are engaged in response to different stimuli will be a challenging goal for further studies.