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Phenotypic Analysis of ATM Protein Kinase in DNA Double-Strand Break Formation and Repair

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ATM Kinase

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1599))

Abstract

Ataxia telangiectasia mutated (ATM) encodes a serine/threonine protein kinase, which is involved in various regulatory processes in mammalian cells. Its best-known role is apical activation of the DNA damage response following generation of DNA double-strand breaks (DSBs). When DSBs appear, sensor and mediator proteins are recruited, activating transducers such as ATM, which in turn relay a widespread signal to a multitude of downstream effectors. ATM mutation causes Ataxia telangiectasia (AT), whereby the disease phenotype shows differing characteristics depending on the underlying ATM mutation. However, all phenotypes share progressive neurodegeneration and marked predisposition to malignancies at the organismal level and sensitivity to ionizing radiation and chromosome aberrations at the cellular level. Expression and localization of the ATM protein can be determined via western blotting and immunofluorescence microscopy; however, detection of subtle alterations such as resulting from amino acid exchanges rather than truncating mutations requires functional testing. Previous studies on the role of ATM in DSB repair, which connects with radiosensitivity and chromosomal stability, gave at first sight contradictory results. To systematically explore the effects of clinically relevant ATM mutations on DSB repair, we engaged a series of lymphoblastoid cell lines (LCLs) derived from AT patients and controls. To examine DSB repair both in a quantitative and qualitative manners, we used an EGFP-based assay comprising different substrates for distinct DSB repair mechanisms. In this way, we demonstrated that particular signaling defects caused by individual ATM mutations led to specific DSB repair phenotypes. To explore the impact of ATM on carcinogenic chromosomal aberrations, we monitored chromosomal breakage at a breakpoint cluster region hotspot within the MLL gene that has been associated with therapy-related leukemia. PCR-based MLL-breakage analysis of HeLa cells treated with and without pharmacological kinase inhibitors revealed ATM-dependent chromatin remodeling at the MLL break site giving access to DNA repair proteins but also nucleases triggering MLL rearrangements. This chapter summarizes these methods for functional characterization of ATM in patient LCLs and human cell lines.

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References

  1. Choi S, Gamper AM, White JS, Bakkenist CJ (2010) Inhibition of ATM kinase activity does not phenocopy ATM protein disruption: implications for the clinical utility of ATM kinase inhibitors. Cell Cycle 9:4052–4057

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. 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

    Article  CAS  Google Scholar 

  3. Lovejoy CA, Cortez D (2009) Common mechanisms of PIKK regulation. DNA Repair 8:1004–1008

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Lempiäinen H, Halazonetis TD (2009) Emerging common themes in regulation of PIKKs and PI3Ks. EMBO J 28:3067–3073

    Article  PubMed  PubMed Central  Google Scholar 

  5. Kim ST, Lim DS, Canmann C, Kastan MB (1999) Substrate specificities and identification of ATM kinase family members. J Biol Chem 274:37538–37543

    Article  CAS  PubMed  Google Scholar 

  6. O'Neill T, Dwyer AJ, Ziv Y, Chan DW, Lees-Miller SP, Abraham RH, Lai JH, Hill D, Shiloh Y, Cantley LC, Rathbun GA (2000) Utilization of oriented peptide libraries to identify substrate motifs selected by ATM. J Biol Chem 275:22719–22727

    Article  PubMed  Google Scholar 

  7. Stiff T, Walker SA, Cerosaletti K, Goodarzi AA, Petermann E, Concannon P, O'Driscoll M, Jeggo PA (2006) ATR-dependent phosphorylation and activation of ATM in response to UV treatment or replication fork stalling. EMBO J 25:5775–5782

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Bakkenist CJ, Kastan MB (2003) DNA damage activates ATM through intermolecular autophosphorylation and dimer dissociation. Nature 421:499–506

    Article  CAS  PubMed  Google Scholar 

  9. White JS, Choi S, Bakkenist CJ (2008) Irreversible chromosome damage accumulates rapidly in the absence of ATM kinase activity. Cell Cycle 7:1277–1284

    Article  CAS  PubMed  Google Scholar 

  10. Kozlov SV, Graham ME, Peng C, Chen P, Robinson PJ, Lavin MF (2006) Involvement of novel autophosphorylation sites in ATM activation. EMBO J 25:3504–3514

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Kozlov SV, Graham ME, Jakob B, Tobias F, Kijas AW, Tanuji M, Chen P, Robinson PJ, Taucher-Scholz G, Suzuki K, So S, Chen D, Lavin MF (2011) Autophosphorylation and ATM activation: additional sites add to the complexity. J Biol Chem 286:9107–9119

    Article  CAS  PubMed  Google Scholar 

  12. Andegeko Y, Moyal L, Mittelman L, Tsarfaty I, Shiloh Y, Rotman G (2001) Nuclear retention of ATM at sites of DNA double strand breaks. J Biol Chem 276:38224–38230

    CAS  PubMed  Google Scholar 

  13. McKinnon PJ (2004) ATM and ataxia telangiectasia. EMBO Rep 5:772–776

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Lavin MF (2008) Ataxia-telangiectasia: from a rare disorder to a paradigm for cell signalling and cancer. Nat Rev Mol Cell Biol 9:759–769

    Article  CAS  PubMed  Google Scholar 

  15. Gilad S, Chessa L, Khosravi R, Russell P, Galanty Y, Piane M, Gatti RA, Jorgensen TJ, Shiloh Y, Bar-Shira A (1998) Genotype-phenotype relationships in ataxia-telangiectasia and variants. Am J Hum Genet 62:551–561

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Keimling M, Volcic M, Csernok A, Wieland B, Dörk T, Wiesmüller L (2011) Functional characterization connects individual patient mutations in ataxia telangiectasia mutated (ATM) with dysfunction of specific DNA double-strand break-repair signaling pathways. FASEB J 25:3849–3860

    Article  CAS  PubMed  Google Scholar 

  17. Renwick A, Thompson D, Seal S, Kelly P, Chagtai T, Ahmed M, North B, Jayatilake H, Barfoot R, Spanova K, McGuffog L, Evans DG, Eccles D, Breast Cancer Susceptibility Collaboration (UK), Easton DF, Stratton MR, Rahman N (2006) ATM mutations that cause ataxia-telangiectasia are breast cancer susceptibility alleles. Nat Genet 38:873–875

    Article  CAS  PubMed  Google Scholar 

  18. Goldgar DE, Healey S, Dowty JG, Da Silva L, Chen X, Spurdle AB, Terry MB, Daly MJ, Buys SM, Southey MC, Andrulis I, John EM, BCFR, kConFab, Khanna KK, Hopper JL, Oefner PJ, Lakhani S, Chenevix-Trench G (2011) Rare variants in the ATM gene and risk of breast cancer. Breast Cancer Res 13:R73

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Lavin MF, Scott S, Gueven N, Kozlov S, Peng C, Chen P (2004) Functional consequences of sequence alterations in the ATM gene. DNA Repair (Amst) 3:1197–1205

    Article  CAS  Google Scholar 

  20. Tomimatsu N, Mukherjee B, Burma S (2009) Distinct roles of ATR and DNA-PKcs in triggering DNA damage responses in ATM-deficient cells. EMBO Rep 10:629–635

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Stewart GS, Maser RS, Stankovic T, Bressan DA, Kaplan MI, Jaspers NG, Raams A, Byrd PJ, Petrini JH, Taylor AM (1999) The DNA double-strand break repair gene hMRE11 is mutated in individuals with an ataxia-telangiectasia-like disorder. Cell 99:577–587

    Article  CAS  PubMed  Google Scholar 

  22. Carney JP, Maser RS, Olivares H, Davis EM, Le Beau M, Yates JR 3rd, Hays L, Morgan WF, Petrini JH (1998) The hMre11/hRad50 protein complex and Nijmegen breakage syndrome: linkage of double-strand break repair to the cellular DNA damage response. Cell 93:477–486

    Article  CAS  PubMed  Google Scholar 

  23. Waltes R, Kalb R, Gatei M, Kijas AW, Stumm M, Sobeck A, Wieland B, Varon R, Lerenthal Y, Lavin MF, Schindler D, Dörk T (2009) Human RAD50 deficiency in a Nijmegen breakage syndrome-like disorder. Am J Hum Genet 84:605–616

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. You Z, Bailis JM, Johnson SA, Dilworth SM, Hunter T (2007) Rapid activation of ATM on DNA flanking double-strand breaks. Nat Cell Biol 9:1311–1318

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Stucki M, Clapperton JA, Mohammad D, Yaffe MB, Smerdon SJ, Jackson SP (2005) MDC1 directly binds phosphorylated histone H2AX to regulate cellular responses to DNA double-strand breaks. Cell 123:1213–1226

    Article  CAS  PubMed  Google Scholar 

  27. Savic V, Yin B, Maas NL, Bredemeyer AL, Carpenter AC, Helmink BA, Yang-Iott KS, Sleckman BP, Bassing CH (2009) Formation of dynamic gamma-H2AX domains along broken DNA strands is distinctly regulated by ATM and MDC1 and dependent upon H2AX densities in chromatin. Mol Cell 34:298–310

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Luo K, Yuan J, Lou Z (2011) Oligomerization of MDC1 protein is important for proper DNA damage response. J Biol Chem 286:28192–28199

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Liu J, Luo S, Zhao H, Liao J, Li J, Yang C, Xu B, Stern DF, Xu X, Ye K (2012) Structural mechanism of the phosphorylation-dependent dimerization of the MDC1 forkhead-associated domain. Nucleic Acids Res 40:3898–3912

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Kim JA, Kruhlak M, Dotiwala F, Nussenzweig A, Haber JE (2007) Heterochromatin is refractory to gamma-H2AX modification in yeast and mammals. J Cell Biol 178:209–218

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Cowell IG, Sunter NJ, Singh PB, Austin CA, Durkacz BW, Tilby MJ (2007) GammaH2AX foci form preferentially in euchromatin after ionising-radiation. PLoS One 2:e1057

    Article  PubMed  PubMed Central  Google Scholar 

  32. 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

    Article  CAS  PubMed  Google Scholar 

  33. Moyal L, Lerenthal Y, Gana-Weisz M, Mass G, So S, Wang SY, Eppink B, Chung YM, Shalev G, Shema E, Shkedy D, Smorodinsky NI, van Vliet N, Kuster B, Mann M, Ciechanover A, Dahm-Daphi J, Kanaar R, Hu MC, Chen DJ, Oren M, Shiloh Y (2011) Requirement of ATM-dependent monoubiquitylation of histone H2B for timely repair of DNA double-strand breaks. Mol Cell 41:529–542

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Gole B, Baumann C, Mian E, Ireno CI, Wiesmüller L (2015) Endonuclease G initiates DNA rearrangements at the MLL breakpoint cluster upon replication stress. Oncogene 34:3391–3401

    Article  CAS  PubMed  Google Scholar 

  35. Robison JG, Elliott J, Dixon K, Oakley GG (2004) Replication protein A and the Mre11.Rad50.Nbs1 complex co-localize and interact at sites of stalled replication forks. J Biol Chem 279:34802–34810

    Article  CAS  PubMed  Google Scholar 

  36. Cannon B, Kuhnlein J, Yang SH, Cheng A, Schindler D, Stark JM, Russell R, Paull TT (2013) Visualization of local DNA unwinding by Mre11/Rad50/Nbs1 using single-molecule FRET. Proc Natl Acad Sci U S A 110:18868–18873

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Shibata A, Moiani D, Arvai AS, Perry J, Harding SM, Genois MM, Maity R, van Rossum-Fikkert S, Kertokalio A, Romoli F, Ismail A, Ismalaj E, Petricci E, Neale MJ, Bristow RG, Masson JY, Wyman C, Jeggo PA, Tainer JA (2014) DNA double-strand break repair pathway choice is directed by distinct MRE11 nuclease activities. Mol Cell 53:7–18

    Article  CAS  PubMed  Google Scholar 

  38. Symington LS, Gautier J (2011) Double-strand break end resection and repair pathway choice. Annu Rev Genet 45:247–271

    Article  CAS  PubMed  Google Scholar 

  39. Kijas AW, Lim Y, Bolderson E, Cerosaletti K, Gatei M, Jakob B, Tobias F, Taucher-Scholz G, Gueven N, Oakley G, Concannon P, Wolvetang E, Khanna KK, Wiesmüller L, Lavin MF (2015) ATM-dependent phosphorylation of MRE11 controls extent of resection during homology directed repair by signalling through exonuclease 1. Nucleic Acids Res 43:8352–8367

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Matsuoka S, Ballif BA, Smogorzewska A, McDonald ER 3rd, 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

    Article  CAS  PubMed  Google Scholar 

  41. Lim DS, Kim ST, Xu B, Maser RS, Lin J, Petrini JH, Kastan MB (2000) ATM phosphorylates p95/nbs1 in an S-phase checkpoint pathway. Nature 404:613–617

    Article  CAS  PubMed  Google Scholar 

  42. Wen J, Cerosaletti K, Schultz KJ, Wright JA, Concannon P (2013) NBN phosphorylation regulates the accumulation of MRN and ATM at sites of DNA double-strand breaks. Oncogene 32:4448–4456

    Article  CAS  PubMed  Google Scholar 

  43. Gatei M, Jakob B, Chen P, Kijas AW, Becherel OJ, Gueven N, Birrell G, Lee JH, Paull TT, Lerenthal Y, Fazry S, Taucher-Scholz G, Kalb R, Schindler D, Waltes R, Dörk T, Lavin MF (2011) ATM protein-dependent phosphorylation of Rad50 protein regulates DNA repair and cell cycle control. J Biol Chem 286:31542–31556

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. Hunt CR, Pandita RK, Laszlo A, Higashikubo R, Agarwal M, Kitamura T, Gupta A, Rief N, Horikoshi N, Baskaran R, Lee JH, Löbrich M, Paull TT, Roti Roti JL, Pandita TK (2007) Hyperthermia activates a subset of ataxia-telangiectasia mutated effectors independent of DNA strand breaks and heat shock protein 70 status. Cancer Res 67:3010–3017

    Article  CAS  PubMed  Google Scholar 

  45. Bencokova Z, Kaufmann MR, Pires IM, Lecane PS, Giaccia AJ, Hammond EM (2009) ATM activation and signaling under hypoxic conditions. Mol Cell Biol 29:526–537

    Article  CAS  PubMed  Google Scholar 

  46. Resseguie EA, Staversky RJ, Brookes PS, O'Reilly MA (2015) Hyperoxia activates ATM independent from mitochondrial ROS and dysfunction. Redox Biol 5:176–185

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  47. Bakkenist CJ, Kastan MB (2015) Chromatin perturbations during the DNA damage response in higher eukaryotes. DNA Repair (Amst) pii:S1568-7864(15)00177-9

    Google Scholar 

  48. Canman CE, Lim DS, Cimprich KA, Taya Y, Tamai K, Sakaguchi K, Appella E, Kastan MB, Siliciano JD (1998) Activation of the ATM kinase by ionizing radiation and phosphorylation of p53. Science 281:1677–1679

    Article  CAS  PubMed  Google Scholar 

  49. Banin S, Moyal L, Shieh S, Taya Y, Anderson CW, Chessa L, Smorodinsky NI, Prives C, Reiss Y, Shiloh Y, Ziv Y (1988) Enhanced phosphorylation of p53 by ATM in response to DNA damage. Science 281:1674–1677

    Article  Google Scholar 

  50. Rashi-Elkeles S, Elkon R, Shavit S, Lerenthal Y, Linhart C, Kupershtein A, Amariglio N, Rechavi G, Shamir R, Shiloh Y (2011) Transcriptional modulation induced by ionizing radiation: p53 remains a central player. Mol Oncol 5:336–348

    Article  CAS  PubMed  Google Scholar 

  51. Gatz SA, Wiesmüller L (2006) p53 in recombination and repair. Cell Death Differ 13:1003–1016

    Article  CAS  PubMed  Google Scholar 

  52. Hadian K, Krappmann D (2011) Signals from the nucleus: activation of NF-kappaB by cytosolic ATM in the DNA damage response. Sci Signal 4:ep2

    Article  Google Scholar 

  53. Levy-Barda A, Lerenthal Y, Davis AJ, Chung YM, Essers J, Shao Z, van Vliet N, Chen DJ, Hu MC, Kanaar R, Ziv Y, Shiloh Y (2011) Involvement of the nuclear proteasome activator PA28γ in the cellular response to DNA double-strand breaks. Cell Cycle 10:4300–4310

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  54. Akyüz N, Boehden GS, Süsse S, Rimek A, Preuss U, Scheidtmann KH, Wiesmüller L (2002) DNA substrate dependence of p53-mediated regulation of double-strand break repair. Mol Cell Biol 22:6306–6317

    Article  PubMed  PubMed Central  Google Scholar 

  55. Kraft D, Rall M, Volcic M, Metzler E, Groo A, Stahl A, Bauer L, Nasonova E, Salles D, Taucher-Scholz G, Bönig H, Fournier C, Wiesmüller L (2015) NF-κB-dependent DNA damage-signaling differentially regulates DNA double-strand break repair mechanisms in immature and mature human hematopoietic cells. Leukemia 29:1543–1554

    Article  CAS  PubMed  Google Scholar 

  56. Obermeier K, Sachsenweger J, Friedl TWP, Pospiech H, Winqvist R, Wiesmüller L (2016) Heterozygous PALB2 c.1592delT mutation channels DNA double-strand break repair into error-prone pathways in breast cancer patients. Oncogene 35(29):3796–3806. doi:10.1038/onc.2015.448

    Article  CAS  PubMed  Google Scholar 

  57. Neitzel H (1986) A routine method for the establishment of permanent growing lymphoblastoid cell lines. Hum Genet 73:320–326

    Article  CAS  PubMed  Google Scholar 

  58. Sandoval N, Platzer M, Rosenthal A, Dörk T, Bendix R, Skawran B, Stuhrmann M, Wegner RD, Sperling K, Banin S, Shiloh Y, Baumer A, Bernthaler U, Sennefelder H, Brohm M, Weber BH, Schindler D (1999) Characterization of ATM gene mutations in 66 ataxia telangiectasia families. Hum Mol Genet 8:69–79

    Article  CAS  PubMed  Google Scholar 

  59. Dörk T, Bendix R, Bremer M, Rades D, Klöpper K, Nicke M, Skawran B, Hector A, Yamini P, Steinmann D, Weise S, Stuhrmann M, Karstens JH (2011) Spectrum of ATM gene mutations in a hospital-based series of unselected breast cancer patients. Cancer Res 61:7608–7615

    Google Scholar 

  60. Keimling M, Kaur J, Bagadi SA, Kreienberg R, Wiesmüller L, Ralhan R (2008) A sensitive test for the detection of specific DSB repair defects in primary cells from breast cancer specimens. Int J Cancer 123:730–736

    Article  CAS  PubMed  Google Scholar 

  61. Speit G, Trenz K, Schütz P, Bendix R, Dörk T (2000) Mutagen sensitivity of human lymphoblastoid cells with a BRCA1 mutation in comparison to ataxia telangiectasia heterozygote cells. Cytogenet Cell Genet 91:261–266

    Article  CAS  PubMed  Google Scholar 

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Authors and Affiliations

  1. Department of Obstetrics and Gynaecology, The University of Ulm, Prittwitzstrasse 43, 89075, Ulm, Germany

    Elisabeth Mian & Lisa Wiesmüller

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  1. Elisabeth Mian
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  2. Lisa Wiesmüller
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Correspondence to Lisa Wiesmüller .

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  1. University of Queensland Centre for Clinical Research (UQCCR), University of Queensland, Herston, Queensland, Australia

    Sergei V. Kozlov

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Mian, E., Wiesmüller, L. (2017). Phenotypic Analysis of ATM Protein Kinase in DNA Double-Strand Break Formation and Repair. In: Kozlov, S. (eds) ATM Kinase. Methods in Molecular Biology, vol 1599. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-6955-5_23

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  • DOI: https://doi.org/10.1007/978-1-4939-6955-5_23

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