Distinct Phosphatases Mediate the Deactivation of the DNA Damage Checkpoint Kinase Rad53

Abstract

The DNA damage checkpoint regulates DNA replication and arrests cell cycle progression in response to genotoxic stress. In Saccharomyces cerevisiae, the protein kinase Rad53 plays a central role in preventing genomic instability and maintaining viability in the presence of replication stress and DNA damage. Activation of Rad53 depends on phosphorylation by the upstream kinase Mec1, followed by autophosphorylation on multiple residues. Also critical for cell viability, the molecular mechanism of Rad53 deactivation remains incompletely understood. Rad53 dephosphorylation after repair of a persistent double strand break in G 2 /M has been shown to depend on the presence of the PP2C-type phosphatases Ptc2 and Ptc3. More recently, the PP2A-like protein phosphatase Pph3 has been shown to be required to dephosphorylate Rad53 after DNA methylation damage in S phase. However, we show here that Ptc2/3 are dispensable for Rad53 deactivation after replication stress or DNA methylation damage. Pph3 is also dispensable for the deactivation of Rad53 after replication stress. In addition, Rad53 kinase activity is still deactivated in pph3 null cells after DNA methylation damage, despite persistent Rad53 hyperphosphorylation. Finally, a strain in which the three phosphatases are deleted shows a severe defect in Rad53 kinase deactivation after DNA methylation damage but not after replication stress. In all, our results suggest that distinct phosphatases operate to return Rad53 to its basal state after different genotoxic stresses and that a yet unidentified phosphatase may be responsible for the deactivation of Rad53 after replication stress.

Dephosphorylation of γH2A by Glc7/Protein Phosphatase 1 Promotes Recovery from Inhibition of DNA Replication

“…The protein phosphatases Pph3, Ptc2, and Ptc3 appear to be dispensable for Rad53 deactivation following HU-induced S phase arrest, while they allow Rad53 deactivation and replication fork restart after MMS exposure (26,32,35). Although Rad53 dephosphorylation is delayed in HU-treated glc7 mutants, it still takes place, raising the possibility that Pph3, Ptc2, and/or Ptc3 might be engaged in cells crippled for Glc7 activity.…”

“…30,2010 Glc7 AND CHECKPOINT INACTIVATION 141 on May 9, 2018 by guest http://mcb.asm.org/ ery after HU-induced fork stalling. According to previous studies (35), we found that pph3⌬ and ptc2⌬ ptc3⌬ cells, which displayed a slight sensitivity to MMS that was enhanced in the triple mutant pph3⌬ ptc2⌬ ptc3⌬, were not more sensitive to HU than wild-type cells (Fig. 8A).…”

“…Furthermore, Pph3/PP4 is required for ␥H2A dephosphorylation, which contributes to recovery from a DSB-induced checkpoint (4,17,25). Finally, Pph3 and Ptc2 have been implicated in Rad53 dephosphorylation and hence deactivation during recovery from MMS exposure (26,32).Notably, none of the above phosphatases is required for Rad53 deactivation after inhibition of DNA replication by deoxynucleoside triphosphate (dNTP) depletion with hydroxyurea (HU), as pph3⌬ ptc2⌬ ptc3⌬ triple mutant cells are not defective in recovering from HU-induced S phase arrest and do not show hypersensitivity to HU treatment (32,35). Thus, a yet-unidentified phosphatase might be responsible for checkpoint termination under these conditions.…”

“…Notably, none of the above phosphatases is required for Rad53 deactivation after inhibition of DNA replication by deoxynucleoside triphosphate (dNTP) depletion with hydroxyurea (HU), as pph3⌬ ptc2⌬ ptc3⌬ triple mutant cells are not defective in recovering from HU-induced S phase arrest and do not show hypersensitivity to HU treatment (32,35). Thus, a yet-unidentified phosphatase might be responsible for checkpoint termination under these conditions.…”

Chromatin Remodeling Factors Isw2 and Ino80 Regulate Checkpoint Activity and Chromatin Structure in S Phase

“…This result demonstrates that proper control of the amplitude and inactivation of the S-phase checkpoint is also essential for DNA replication control in the presence of replication stress. It also has been shown that other modes of checkpoint deactivation exist that have yet to be identified, highlighting the need for more studies on checkpoint deactivation mechanisms (Travesa et al 2008).…”