title: Yeast PP4 interacts with ATR homolog Ddc2-Mec1 and regulates checkpoint signaling.
abstract: Mec1-Ddc2 (ATR-ATRIP) controls the DNA damage checkpoint and shows differential cell-cycle regulation in yeast. To find regulators of Mec1-Ddc2, we exploited a mec1 mutant that retains catalytic activity in G2 and recruitment to stalled replication forks, but which is compromised for the intra-S phase checkpoint. Two screens, one for spontaneous survivors and an E-MAP screen for synthetic growth effects, identified loss of PP4 phosphatase, pph3Δ and psy2Δ, as the strongest suppressors of mec1-100 lethality on HU. Restored Rad53 phosphorylation accounts for part, but not all, of the pph3Δ-mediated survival. Phosphoproteomic analysis confirmed that 94% of the mec1-100-compromised targets on HU are PP4 regulated, including a phosphoacceptor site within Mec1 itself, mutation of which confers damage sensitivity. Physical interaction between Pph3 and Mec1, mediated by cofactors Psy2 and Ddc2, is shown biochemically and through FRET in subnuclear repair foci. This establishes a physical and functional Mec1-PP4 unit for regulating the checkpoint response.
authors.affiliation: Friedrich Miescher Institute for Biomedical Research, Maulbeerstrasse 66, 4058 Basel, Switzerland; Faculty of Sciences, University of Basel, 4056 Basel, Switzerland.
authors.forename: Nicole
authors.initials: N
authors.lastname: Hustedt
authors.affiliation: Friedrich Miescher Institute for Biomedical Research, Maulbeerstrasse 66, 4058 Basel, Switzerland; Faculty of Sciences, University of Basel, 4056 Basel, Switzerland.
authors.forename: Andrew
authors.initials: A
authors.lastname: Seeber
authors.affiliation: Friedrich Miescher Institute for Biomedical Research, Maulbeerstrasse 66, 4058 Basel, Switzerland.
authors.forename: Ragna
authors.initials: R
authors.lastname: Sack
authors.affiliation: Friedrich Miescher Institute for Biomedical Research, Maulbeerstrasse 66, 4058 Basel, Switzerland.
authors.forename: Monika
authors.initials: M
authors.lastname: Tsai-Pflugfelder
authors.affiliation: Novartis Institutes for Biomedical Research, Novartis Pharma AG, Fabrikstrasse 22, 4056 Basel, Switzerland.
authors.forename: Bhupinder
authors.initials: B
authors.lastname: Bhullar
authors.affiliation: Division of Gene Regulation, The Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX Amsterdam, the Netherlands.
authors.forename: Hanneke
authors.initials: H
authors.lastname: Vlaming
authors.affiliation: Division of Gene Regulation, The Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX Amsterdam, the Netherlands.
authors.forename: Fred
authors.initials: F
authors.lastname: van Leeuwen
authors.affiliation: Department of Toxicogenetics, Leiden University Medical Center, Einthovenweg 20, 2333 ZC Leiden, the Netherlands.
authors.forename: Aude
authors.initials: A
authors.lastname: Guénolé
authors.affiliation: Department of Toxicogenetics, Leiden University Medical Center, Einthovenweg 20, 2333 ZC Leiden, the Netherlands.
authors.forename: Haico
authors.initials: H
authors.lastname: van Attikum
authors.affiliation: Departments of Bioengineering and Medicine, University of California, San Diego, La Jolla, CA 92093, USA.
authors.forename: Rohith
authors.initials: R
authors.lastname: Srivas
authors.affiliation: Departments of Bioengineering and Medicine, University of California, San Diego, La Jolla, CA 92093, USA.
authors.forename: Trey
authors.initials: T
authors.lastname: Ideker
authors.affiliation: Friedrich Miescher Institute for Biomedical Research, Maulbeerstrasse 66, 4058 Basel, Switzerland.
authors.forename: Kenji
authors.initials: K
authors.lastname: Shimada
authors.affiliation: Friedrich Miescher Institute for Biomedical Research, Maulbeerstrasse 66, 4058 Basel, Switzerland; Faculty of Sciences, University of Basel, 4056 Basel, Switzerland. Electronic address: susan.gasser@fmi.ch.
authors.forename: Susan M
authors.initials: SM
authors.lastname: Gasser
mesh_terms: D048868:Adaptor Proteins, Signal Transducing / Q000378:metabolism*; D059447:Cell Cycle Checkpoints; D018797:Cell Cycle Proteins / Q000378:metabolism*; D064447:Checkpoint Kinase 2 / Q000378:metabolism; D004261:DNA Replication; D004843:Epistasis, Genetic; D015966:Gene Expression Regulation, Fungal; D057809:HEK293 Cells; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000378:metabolism*; D009687:Nuclear Proteins / Q000378:metabolism*; D010749:Phosphoprotein Phosphatases / Q000378:metabolism*; D010766:Phosphorylation; D011499:Protein Processing, Post-Translational; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000166:cytology / Q000201:enzymology*; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism*; D015398:Signal Transduction
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D018797:Cell Cycle Proteins; D047908:Intracellular Signaling Peptides and Proteins; C440014:LCD1 protein, S cerevisiae; D009687:Nuclear Proteins; C494616:Psy2 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D064447:Checkpoint Kinase 2; C088937:MEC1 protein, S cerevisiae; D017346:Protein Serine-Threonine Kinases; C067022:RAD53 protein, S cerevisiae; C502440:PPH3 protein, S cerevisiae; D010749:Phosphoprotein Phosphatases