Distinct phosphatases mediate the deactivation of the DNA damage checkpoint kinase Rad53.
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.
Travesa
Anna
A
Department of Biochemistry and Molecular Biology, School of Medicine, and the Center for Biophysical Studies, Universitat Autonoma de Barcelona, Bellaterra, Catalonia, Spain.
Duch
Alba
A
Quintana
David G
DG
D002453:Cell Cycle; D018797:Cell Cycle Proteins / Q000378:metabolism*; D064447:Checkpoint Kinase 2; D004249:DNA Damage; D019175:DNA Methylation; D004261:DNA Replication; D015971:Gene Expression Regulation, Enzymologic; D015966:Gene Expression Regulation, Fungal; D009154:Mutation; D010749:Phosphoprotein Phosphatases / Q000378:metabolism; D010744:Phosphoric Monoester Hydrolases / Q000378:metabolism; D010766:Phosphorylation; D054648:Protein Phosphatase 2; D000071636:Protein Phosphatase 2C; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D016196:S Phase; D012441:Saccharomyces cerevisiae / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism*
D018797:Cell Cycle Proteins; D029701:Saccharomyces cerevisiae Proteins; D064447:Checkpoint Kinase 2; D017346:Protein Serine-Threonine Kinases; C067022:RAD53 protein, S cerevisiae; C502440:PPH3 protein, S cerevisiae; C491716:PTC2 protein, S cerevisiae; C000607464:PTC3 protein, S cerevisiae; D010749:Phosphoprotein Phosphatases; D054648:Protein Phosphatase 2; D000071636:Protein Phosphatase 2C; D010744:Phosphoric Monoester Hydrolases