title: Termination of Replication Stress Signaling via Concerted Action of the Slx4 Scaffold and the PP4 Phosphatase.
abstract: In response to replication stress, signaling mediated by DNA damage checkpoint kinases protects genome integrity. However, following repair or bypass of DNA lesions, checkpoint signaling needs to be terminated for continued cell cycle progression and proliferation. In budding yeast, the PP4 phosphatase has been shown to play a key role in preventing hyperactivation of the checkpoint kinase Rad53. In addition, we recently uncovered a phosphatase-independent mechanism for downregulating Rad53 in which the DNA repair scaffold Slx4 decreases engagement of the checkpoint adaptor Rad9 at DNA lesions. Here we reveal that proper termination of checkpoint signaling following the bypass of replication blocks imposed by alkylated DNA adducts requires the concerted action of these two fundamentally distinct mechanisms of checkpoint downregulation. Cells lacking both SLX4 and the PP4-subunit PPH3 display a synergistic increase in Rad53 signaling and are exquisitely sensitive to the DNA alkylating agent methyl methanesulfonate, which induces replication blocks and extensive formation of chromosomal linkages due to template switching mechanisms required for fork bypass. Rad53 hypersignaling in these cells seems to converge to a strong repression of Mus81-Mms4, the endonuclease complex responsible for resolving chromosomal linkages, thus explaining the selective sensitivity of slx4Δ pph3Δ cells to alkylation damage. Our results support a model in which Slx4 acts locally to downregulate Rad53 activation following fork bypass, while PP4 acts on pools of active Rad53 that have diffused from the site of lesions. We propose that the proper spatial coordination of the Slx4 scaffold and PP4 action is crucial to allow timely activation of Mus81-Mms4 and, therefore, proper chromosome segregation.
authors.affiliation: Department of Molecular Biology and Genetics, Weill Institute for Cell and Molecular Biology, Cornell University, Ithaca, New York 14853.
authors.forename: Carolyn M
authors.initials: CM
authors.lastname: Jablonowski
authors.affiliation: Department of Molecular Biology and Genetics, Weill Institute for Cell and Molecular Biology, Cornell University, Ithaca, New York 14853.
authors.forename: José R
authors.identifier: http://orcid.org/0000-0001-5909-8215
authors.initials: JR
authors.lastname: Cussiol
authors.affiliation: Department of Molecular Biology and Genetics, Weill Institute for Cell and Molecular Biology, Cornell University, Ithaca, New York 14853.
authors.forename: Susannah
authors.initials: S
authors.lastname: Oberly
authors.affiliation: Donnelly Centre and Department of Biochemistry, University of Toronto, Toronto, Ontario, M5S 3E1, Canada.
authors.forename: Askar
authors.initials: A
authors.lastname: Yimit
authors.affiliation: Donnelly Centre and Department of Biochemistry, University of Toronto, Toronto, Ontario, M5S 3E1, Canada.
authors.forename: Attila
authors.initials: A
authors.lastname: Balint
authors.affiliation: Donnelly Centre and Department of Computer Science, University of Toronto, Toronto, Ontario, M5S 3E1, Canada.
authors.forename: TaeHyung
authors.initials: T
authors.lastname: Kim
authors.affiliation: Donnelly Centre and Department of Computer Science, University of Toronto, Toronto, Ontario, M5S 3E1, Canada.
authors.forename: Zhaolei
authors.initials: Z
authors.lastname: Zhang
authors.affiliation: Donnelly Centre and Department of Computer Science, University of Toronto, Toronto, Ontario, M5S 3E1, Canada.
authors.forename: Grant W
authors.initials: GW
authors.lastname: Brown
authors.affiliation: Department of Molecular Biology and Genetics, Weill Institute for Cell and Molecular Biology, Cornell University, Ithaca, New York 14853 mbs266@cornell.edu.
authors.forename: Marcus B
authors.initials: MB
authors.lastname: Smolka
mesh_terms: D059447:Cell Cycle Checkpoints; D018797:Cell Cycle Proteins / Q000378:metabolism; D064447:Checkpoint Kinase 2 / Q000378:metabolism; D018736:DNA Adducts / Q000378:metabolism; D004249:DNA Damage; D004261:DNA Replication; D004271:DNA, Fungal / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism; D015536:Down-Regulation; D004706:Endodeoxyribonucleases / Q000378:metabolism*; D004720:Endonucleases / Q000378:metabolism; D045585:Flap Endonucleases / Q000378:metabolism; D009687:Nuclear Proteins / Q000378:metabolism; D010749:Phosphoprotein Phosphatases / Q000378:metabolism; D010766:Phosphorylation; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism*; D015398:Signal Transduction
chemical_list: D018797:Cell Cycle Proteins; D018736:DNA Adducts; D004271:DNA, Fungal; D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; C501265:RTT107 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D064447:Checkpoint Kinase 2; C067022:RAD53 protein, S cerevisiae; D004706:Endodeoxyribonucleases; D004720:Endonucleases; D045585:Flap Endonucleases; C411624:MUS81 protein, S cerevisiae; C505436:SLX4 protein, S cerevisiae; C112337:MMS4 protein, S cerevisiae; C502440:PPH3 protein, S cerevisiae; D010749:Phosphoprotein Phosphatases
keywords: DNA damage checkpoint; PP4; Rad53; Slx4; replication stress