title: Pph3-Psy2 is a phosphatase complex required for Rad53 dephosphorylation and replication fork restart during recovery from DNA damage.
abstract: Activation of the checkpoint kinase Rad53 is a critical response to DNA damage that results in stabilization of stalled replication forks, inhibition of late-origin initiation, up-regulation of dNTP levels, and delayed entry to mitosis. Activation of Rad53 is well understood and involves phosphorylation by the protein kinases Mec1 and Tel1 as well as in trans autophosphorylation by Rad53 itself. However, deactivation of Rad53, which must occur to allow the cell to recover from checkpoint arrest, is not well understood. Here, we present genetic and biochemical evidence that the type 2A-like protein phosphatase Pph3 forms a complex with Psy2 (Pph3-Psy2) that binds and dephosphorylates activated Rad53 during treatment with, and recovery from, methylmethane sulfonate-mediated DNA damage. In the absence of Pph3-Psy2, Rad53 dephosphorylation and the resumption of DNA synthesis are delayed during recovery from DNA damage. This delay in DNA synthesis reflects a failure to restart stalled replication forks, whereas, remarkably, genome replication is eventually completed by initiating late origins of replication despite the presence of hyperphosphorylated Rad53. These findings suggest that Rad53 regulates replication fork restart and initiation of late firing origins independently and that regulation of these processes is mediated by specific Rad53 phosphatases.
authors.affiliation: Departments of Chemistry and Cell Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA.
authors.forename: Bryan M
authors.initials: BM
authors.lastname: O'Neill
authors.forename: Shawn J
authors.initials: SJ
authors.lastname: Szyjka
authors.forename: Ewa T
authors.initials: ET
authors.lastname: Lis
authors.forename: Aaron O
authors.initials: AO
authors.lastname: Bailey
authors.forename: John R
authors.initials: JR
authors.lastname: Yates
authors.forename: Oscar M
authors.initials: OM
authors.lastname: Aparicio
authors.forename: Floyd E
authors.initials: FE
authors.lastname: Romesberg
mesh_terms: D018797:Cell Cycle Proteins / Q000235:genetics / Q000378:metabolism*; D064447:Checkpoint Kinase 2; D004249:DNA Damage / Q000235:genetics*; D004261:DNA Replication / Q000235:genetics*; D004271:DNA, Fungal / Q000235:genetics* / Q000378:metabolism; D004789:Enzyme Activation; D015966:Gene Expression Regulation, Fungal; D006657:Histones / Q000235:genetics / Q000378:metabolism; D008741:Methyl Methanesulfonate / Q000494:pharmacology; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism*; D010749:Phosphoprotein Phosphatases / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D011485:Protein Binding; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism*
chemical_list: D018797:Cell Cycle Proteins; D004271:DNA, Fungal; D006657:Histones; D009687:Nuclear Proteins; C494616:Psy2 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; C507778:gammaH2AX protein, S cerevisiae; D008741:Methyl Methanesulfonate; D064447:Checkpoint Kinase 2; D017346:Protein Serine-Threonine Kinases; C067022:RAD53 protein, S cerevisiae; C502440:PPH3 protein, S cerevisiae; D010749:Phosphoprotein Phosphatases