After DNA damage, the cell cycle is arrested to avoid propagation of mutations. Arrest in G2 phase is initiated by ATM-/ATR-dependent signaling that inhibits mitosis-promoting kinases such as Plk1. At the same time, Plk1 can counteract ATR-dependent signaling and is required for eventual resumption of the cell cycle. However, what determines when Plk1 activity can resume remains unclear. Here, we use FRET-based reporters to show that a global spread of ATM activity on chromatin and phosphorylation of ATM targets including KAP1 control Plk1 re-activation. These phosphorylations are rapidly counteracted by the chromatin-bound phosphatase Wip1, allowing cell cycle restart despite persistent ATM activity present at DNA lesions. Combining experimental data and mathematical modeling, we propose a model for how the minimal duration of cell cycle arrest is controlled. Our model shows how cell cycle restart can occur before completion of DNA repair and suggests a mechanism for checkpoint adaptation in human cells.

Department of Cell and Molecular Biology Karolinska Institutet Stockholm Sweden

Department of Medical Biochemistry and Biophysics and Science for Life Laboratory Karolinska Institutet Stockholm Sweden

Division of Cell Biology Netherlands Cancer Institute Amsterdam The Netherlands

Faculty of Science Charles University Prague Prague Czech Republic

Laboratory of Cancer Cell Biology Institute of Molecular Genetics Academy of Sciences of the Czech Republic Prague Czech Republic

Zobrazit více v PubMed

Akopyan K, Silva Cascales H, Hukasova E, Saurin AT, Mullers E, Jaiswal H, Hollman DA, Kops GJ, Medema RH, Lindqvist A (2014) Assessing kinetics from fixed cells reveals activation of the mitotic entry network at the S/G2 transition. Mol Cell 53: 843–853 PubMed

Altmeyer M, Lukas J (2013) To spread or not to spread–chromatin modifications in response to DNA damage. Curr Opin Genet Dev 23: 156–165 PubMed

Bartek J, Lukas J (2007) DNA damage checkpoints: from initiation to recovery or adaptation. Curr Opin Cell Biol 19: 238–245 PubMed

Belyi VA, Ak P, Markert E, Wang H, Hu W, Puzio‐Kuter A, Levine AJ (2010) The origins and evolution of the p53 family of genes. Cold Spring Harb Perspect Biol 2: a001198 PubMed PMC

de Boer HR, Guerrero Llobet S, van Vugt MA (2016) Controlling the response to DNA damage by the APC/C‐Cdh1. Cell Mol Life Sci 73: 949–960 PubMed PMC

Bruinsma W, Aprelia M, García‐Santisteban I, Kool J, Xu YJ, Medema RH (2017) Inhibition of Polo‐like kinase 1 during the DNA damage response is mediated through loss of Aurora A recruitment by Bora. Oncogene 36: 1840–1848 PubMed PMC

Bulavin DV, Demidov ON, Saito S, Kauraniemi P, Phillips C, Amundson SA, Ambrosino C, Sauter G, Nebreda AR, Anderson CW, Kallioniemi A, Fornace AJ, Appella E (2002) Amplification of PPM1D in human tumors abrogates p53 tumor‐suppressor activity. Nat Gen 31: 210–215 PubMed

Bunz F, Dutriaux A, Lengauer C, Waldman T, Zhou S, Brown JP, Sedivy JM, Kinzler KW, Vogelstein B (1998) Requirement for p53 and p21 to sustain G2 arrest after DNA damage. Science 282: 1497–1501 PubMed

Charrier JD, Durrant SJ, Golec JM, Kay DP, Knegtel RM, MacCormick S, Mortimore M, O'Donnell ME, Pinder JL, Reaper PM, Rutherford AP, Wang PS, Young SC, Pollard JR (2011) Discovery of potent and selective inhibitors of ataxia telangiectasia mutated and Rad3 related (ATR) protein kinase as potential anticancer agents. J Med Chem 54: 2320–2330 PubMed

Deckbar D, Birraux J, Krempler A, Tchouandong L, Beucher A, Walker S, Stiff T, Jeggo P, Lobrich M (2007) Chromosome breakage after G2 checkpoint release. J Cell Biol 176: 749–755 PubMed PMC

Donnianni RA, Ferrari M, Lazzaro F, Clerici M, Tamilselvan Nachimuthu B, Plevani P, Muzi‐Falconi M, Pellicioli A (2010) Elevated levels of the polo kinase Cdc5 override the Mec1/ATR checkpoint in budding yeast by acting at different steps of the signaling pathway. PLoS Genet 6: e1000763 PubMed PMC

Durocher D, Taylor IA, Sarbassova D, Haire LF, Westcott SL, Jackson SP, Smerdon SJ, Yaffe MB (2000) The molecular basis of FHA domain: phosphopeptide binding specificity and implications for phospho‐dependent signaling mechanisms. Mol Cell 6: 1169–1182 PubMed

Floyd SR, Pacold ME, Huang Q, Clarke SM, Lam FC, Cannell IG, Bryson BD, Rameseder J, Lee MJ, Blake EJ, Fydrych A, Ho R, Greenberger BA, Chen GC, Maffa A, Del Rosario AM, Root DE, Carpenter AE, Hahn WC, Sabatini DM et al (2013) The bromodomain protein Brd4 insulates chromatin from DNA damage signalling. Nature 498: 246–250 PubMed PMC

Fuller BG, Lampson MA, Foley EA, Rosasco‐Nitcher S, Le KV, Tobelmann P, Brautigan DL, Stukenberg PT, Kapoor TM (2008) Midzone activation of aurora B in anaphase produces an intracellular phosphorylation gradient. Nature 453: 1132–1136 PubMed PMC

Hukasova E, Silva Cascales H, Kumar SR, Lindqvist A (2012) Monitoring kinase and phosphatase activities through the cell cycle by ratiometric FRET. J Vis Exp 59: 3410 PubMed PMC

Jackson SP, Bartek J (2009) The DNA‐damage response in human biology and disease. Nature 461: 1071–1078 PubMed PMC

Johnson SA, You Z, Hunter T (2007) Monitoring ATM kinase activity in living cells. DNA Repair 6: 1277–1284 PubMed

Kim BJ, Li Y, Zhang J, Xi Y, Li Y, Yang T, Jung SY, Pan X, Chen R, Li W, Wang Y, Qin J (2010) Genome‐wide reinforcement of cohesin binding at pre‐existing cohesin sites in response to ionizing radiation in human cells. J Biol Chem 285: 22784–22792 PubMed PMC

Kousholt AN, Fugger K, Hoffmann S, Larsen BD, Menzel T, Sartori AA, Sorensen CS (2012) CtIP‐dependent DNA resection is required for DNA damage checkpoint maintenance but not initiation. J Cell Biol 197: 869–876 PubMed PMC

Krenning L, Feringa FM, Shaltiel IA, van den Berg J, Medema RH (2014) Transient activation of p53 in G2 phase is sufficient to induce senescence. Mol Cell 55: 59–72 PubMed

Krystyniak A, Garcia‐Echeverria C, Prigent C, Ferrari S (2006) Inhibition of Aurora A in response to DNA damage. Oncogene 25: 338–348 PubMed

Lee J, Kumagai A, Dunphy WG (2001) Positive regulation of Wee1 by Chk1 and 14‐3‐3 proteins. Mol Biol Cell 12: 551–563 PubMed PMC

Lee DH, Goodarzi AA, Adelmant GO, Pan Y, Jeggo PA, Marto JA, Chowdhury D (2012) Phosphoproteomic analysis reveals that PP4 dephosphorylates KAP‐1 impacting the DNA damage response. EMBO J 31: 2403–2415 PubMed PMC

Li X, Lee YK, Jeng JC, Yen Y, Schultz DC, Shih HM, Ann DK (2007) Role for KAP1 serine 824 phosphorylation and sumoylation/desumoylation switch in regulating KAP1‐mediated transcriptional repression. J Biol Chem 282: 36177–36189 PubMed

Liang Q, Deng H, Li X, Wu X, Tang Q, Chang TH, Peng H, Rauscher FJ III, Ozato K, Zhu F (2011) Tripartite motif‐containing protein 28 is a small ubiquitin‐related modifier E3 ligase and negative regulator of IFN regulatory factor 7. J Immunol 187: 4754–4763 PubMed PMC

Liang H, Esposito A, De S, Ber S, Collin P, Surana U, Venkitaraman AR (2014) Homeostatic control of polo‐like kinase‐1 engenders non‐genetic heterogeneity in G2 checkpoint fidelity and timing. Nat Commun 5: 4048 PubMed PMC

Lindqvist A, de Bruijn M, Macurek L, Bras A, Mensinga A, Bruinsma W, Voets O, Kranenburg O, Medema RH (2009) Wip1 confers G2 checkpoint recovery competence by counteracting p53‐dependent transcriptional repression. EMBO J 28: 3196–3206 PubMed PMC

Lobrich M, Jeggo PA (2007) The impact of a negligent G2/M checkpoint on genomic instability and cancer induction. Nat Rev Cancer 7: 861–869 PubMed

Lock RB, Ross WE (1990) Inhibition of p34cdc2 kinase activity by etoposide or irradiation as a mechanism of G2 arrest in Chinese hamster ovary cells. Cancer Res 50: 3761–3766 PubMed

Loewer A, Karanam K, Mock C, Lahav G (2013) The p53 response in single cells is linearly correlated to the number of DNA breaks without a distinct threshold. BMC Biol 11: 114 PubMed PMC

Macurek L, Lindqvist A, Lim D, Lampson MA, Klompmaker R, Freire R, Clouin C, Taylor SS, Yaffe MB, Medema RH (2008) Polo‐like kinase‐1 is activated by aurora A to promote checkpoint recovery. Nature 455: 119–123 PubMed

Macurek L, Lindqvist A, Medema RH (2009) Aurora‐A and hBora join the game of Polo. Cancer Res 69: 4555–4558 PubMed

Macurek L, Lindqvist A, Voets O, Kool J, Vos HR, Medema RH (2010) Wip1 phosphatase is associated with chromatin and dephosphorylates gammaH2AX to promote checkpoint inhibition. Oncogene 29: 2281–2291 PubMed

Mailand N, Falck J, Lukas C, Syljuasen RG, Welcker M, Bartek J, Lukas J (2000) Rapid destruction of human Cdc25A in response to DNA damage. Science 288: 1425–1429 PubMed

Mailand N, Bekker‐Jensen S, Bartek J, Lukas J (2006) Destruction of Claspin by SCFbetaTrCP restrains Chk1 activation and facilitates recovery from genotoxic stress. Mol Cell 23: 307–318 PubMed

Mamely I, van Vugt MA, Smits VA, Semple JI, Lemmens B, Perrakis A, Medema RH, Freire R (2006) Polo‐like kinase‐1 controls proteasome‐dependent degradation of Claspin during checkpoint recovery. Curr Biol 16: 1950–1955 PubMed

Matsuoka S, Ballif BA, Smogorzewska A, McDonald ER III, Hurov KE, Luo J, Bakalarski CE, Zhao Z, Solimini N, Lerenthal Y, Shiloh Y, Gygi SP, Elledge SJ (2007) ATM and ATR substrate analysis reveals extensive protein networks responsive to DNA damage. Science 316: 1160–1166 PubMed

Medema RH, Macurek L (2011) Checkpoint control and cancer. Oncogene 31: 2601–2613 PubMed

Mu JJ, Wang Y, Luo H, Leng M, Zhang J, Yang T, Besusso D, Jung SY, Qin J (2007) A proteomic analysis of ataxia telangiectasia‐mutated (ATM)/ATM‐Rad3‐related (ATR) substrates identifies the ubiquitin‐proteasome system as a regulator for DNA damage checkpoints. J Biol Chem 282: 17330–17334 PubMed

Mullers E, Silva Cascales H, Jaiswal H, Saurin AT, Lindqvist A (2014) Nuclear translocation of Cyclin B1 marks the restriction point for terminal cell cycle exit in G2 phase. Cell Cycle 13: 2733–2743 PubMed PMC

Munoz DP, Kawahara M, Yannone SM (2013) An autonomous chromatin/DNA‐PK mechanism for localized DNA damage signaling in mammalian cells. Nucleic Acids Res 41: 2894–2906 PubMed PMC

Peng CY, Graves PR, Thoma RS, Wu Z, Shaw AS, Piwnica‐Worms H (1997) Mitotic and G2 checkpoint control: regulation of 14‐3‐3 protein binding by phosphorylation of Cdc25C on serine‐216. Science 277: 1501–1505 PubMed

Peschiaroli A, Dorrello NV, Guardavaccaro D, Venere M, Halazonetis T, Sherman NE, Pagano M (2006) SCFbetaTrCP‐mediated degradation of Claspin regulates recovery from the DNA replication checkpoint response. Mol Cell 23: 319–329 PubMed

Peter M, Nakagawa J, Doree M, Labbe JC, Nigg EA (1990) In vitro disassembly of the nuclear lamina and M phase‐specific phosphorylation of lamins by cdc2 kinase. Cell 61: 591–602 PubMed

Qin B, Gao B, Yu J, Yuan J, Lou Z (2013) Ataxia telangiectasia‐mutated‐ and Rad3‐related protein regulates the DNA damage‐induced G2/M checkpoint through the Aurora A cofactor Bora protein. J Biol Chem 288: 16139–16144 PubMed PMC

Rauta J, Alarmo EL, Kauraniemi P, Karhu R, Kuukasjarvi T, Kallioniemi A (2006) The serine‐threonine protein phosphatase PPM1D is frequently activated through amplification in aggressive primary breast tumours. Breast Cancer Res Treat 95: 257–263 PubMed

Reaper PM, Griffiths MR, Long JM, Charrier JD, Maccormick S, Charlton PA, Golec JM, Pollard JR (2011) Selective killing of ATM‐ or p53‐deficient cancer cells through inhibition of ATR. Nat Chem Biol 7: 428–430 PubMed

Reinhardt HC, Aslanian AS, Lees JA, Yaffe MB (2007) p53‐deficient cells rely on ATM‐ and ATR‐mediated checkpoint signaling through the p38MAPK/MK2 pathway for survival after DNA damage. Cancer Cell 11: 175–189 PubMed PMC

Reinhardt HC, Yaffe MB (2009) Kinases that control the cell cycle in response to DNA damage: Chk1, Chk2, and MK2. Curr Opin Cell Biol 21: 245–255 PubMed PMC

Reinhardt HC, Hasskamp P, Schmedding I, Morandell S, van Vugt MA, Wang X, Linding R, Ong SE, Weaver D, Carr SA, Yaffe MB (2010) DNA damage activates a spatially distinct late cytoplasmic cell‐cycle checkpoint network controlled by MK2‐mediated RNA stabilization. Mol Cell 40: 34–49 PubMed PMC

Sanchez Y, Wong C, Thoma RS, Richman R, Wu Z, Piwnica‐Worms H, Elledge SJ (1997) Conservation of the Chk1 checkpoint pathway in mammals: linkage of DNA damage to Cdk regulation through Cdc25. Science 277: 1497–1501 PubMed

Shiloh Y, Ziv Y (2013) The ATM protein kinase: regulating the cellular response to genotoxic stress, and more. Nat Rev Mol Cell Biol 14: 197–210 PubMed

Shiotani B, Zou L (2009) Single‐stranded DNA orchestrates an ATM‐to‐ATR switch at DNA breaks. Mol Cell 33: 547–558 PubMed PMC

Shreeram S, Demidov ON, Hee WK, Yamaguchi H, Onishi N, Kek C, Timofeev ON, Dudgeon C, Fornace AJ, Anderson CW, Minami Y, Appella E, Bulavin DV (2006) Wip1 phosphatase modulates ATM‐dependent signaling pathways. Mol Cell 23: 757–764 PubMed

Smits VA, Klompmaker R, Arnaud L, Rijksen G, Nigg EA, Medema RH (2000) Polo‐like kinase‐1 is a target of the DNA damage checkpoint. Nat Cell Biol 2: 672–676 PubMed

Syljuasen RG, Jensen S, Bartek J, Lukas J (2006) Adaptation to the ionizing radiation‐induced G2 checkpoint occurs in human cells and depends on checkpoint kinase 1 and Polo‐like kinase 1 kinases. Cancer Res 66: 10253–10257 PubMed

Tkacz‐Stachowska K, Lund‐Andersen C, Velissarou A, Myklebust JH, Stokke T, Syljuasen RG (2011) The amount of DNA damage needed to activate the radiation‐induced G2 checkpoint varies between single cells. Radiother Oncol 101: 24–27 PubMed

Toczyski DP, Galgoczy DJ, Hartwell LH (1997) CDC5 and CKII control adaptation to the yeast DNA damage checkpoint. Cell 90: 1097–1106 PubMed

van Vugt MA, Bras A, Medema RH (2004) Polo‐like kinase‐1 controls recovery from a G2 DNA damage‐induced arrest in mammalian cells. Mol Cell 15: 799–811 PubMed

van Vugt MA, Gardino AK, Linding R, Ostheimer GJ, Reinhardt HC, Ong SE, Tan CS, Miao H, Keezer SM, Li J, Pawson T, Lewis TA, Carr SA, Smerdon SJ, Brummelkamp TR, Yaffe MB (2010) A mitotic phosphorylation feedback network connects Cdk1, Plk 1, 53BP1, and Chk2 to inactivate the G(2)/M DNA damage checkpoint. PLoS Biol 8: e1000287 PubMed PMC

Wang L, Guo Q, Fisher LA, Liu D, Peng A (2015) Regulation of polo‐like kinase 1 by DNA damage and PP2A/B55alpha. Cell Cycle 14: 157–166 PubMed PMC

Yamaguchi H, Durell SR, Chatterjee DK, Anderson CW, Appella E (2007) The Wip1 phosphatase PPM1D dephosphorylates SQ/TQ motifs in checkpoint substrates phosphorylated by PI3K‐like kinases. Biochemistry 46: 12594–12603 PubMed

Ziv Y, Bielopolski D, Galanty Y, Lukas C, Taya Y, Schultz DC, Lukas J, Bekker‐Jensen S, Bartek J, Shiloh Y (2006) Chromatin relaxation in response to DNA double‐strand breaks is modulated by a novel ATM‐ and KAP‐1 dependent pathway. Nat Cell Biol 8: 870–876 PubMed

PPM1D activity promotes cellular transformation by preventing senescence and cell death

PPM1D activity promotes the replication stress caused by cyclin E1 overexpression

Phosphorylation of TRF2 promotes its interaction with TIN2 and regulates DNA damage response at telomeres

Truncated PPM1D Prevents Apoptosis in the Murine Thymus and Promotes Ionizing Radiation-Induced Lymphoma

A comprehensive evaluation of pathogenic mutations in primary cutaneous melanomas, including the identification of novel loss-of-function variants

Truncated PPM1D impairs stem cell response to genotoxic stress and promotes growth of APC-deficient tumors in the mouse colon

WIP1 Promotes Homologous Recombination and Modulates Sensitivity to PARP Inhibitors