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ATM Kinase pp 287-302 | Cite as

Live Cell Imaging to Study Real-Time ATM-Mediated Recruitment of DNA Repair Complexes to Sites of Ionizing Radiation-Induced DNA Damage

  • Burkhard JakobEmail author
  • Gisela Taucher-Scholz
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1599)

Abstract

Measurements of protein recruitment and the formation of repair complexes at DNA double-strand breaks in real time provide valuable insight into the regulation of the early DNA damage response. Here, we describe the use of live cell microscopy in combination with ionizing radiation as a tool to evaluate the influence of ATM and its site-specific phosphorylation of target proteins on these processes. Recommendations are made for the preparation of the cells and the design of specialized cell chambers for the localized (and/or targeted) irradiation with charged particles at accelerator beamlines as well as the microscopic equipment and protocol to obtain high-resolution, sensitive fluorescence measurements.

Key words

DNA damage Double strand breaks (DSB) ATM Live-imaging Protein recruitment GFP Microscopy 

Notes

Acknowledgment

This work was supported by BMBF Grants 02NUK037A, 02NUK001A and DFG GRK1657.

References

  1. 1.
    Shiloh Y (2014) ATM: expanding roles as a chief guardian of genome stability. Exp Cell Res 329(1):154–161CrossRefPubMedGoogle Scholar
  2. 2.
    Rogakou EP, Pilch DR, Orr AH, Ivanova VS, Bonner WM (1998) DNA double-stranded breaks induce histone H2AX phosphorylation on serine 139. J Biol Chem 273(10):5858–5868CrossRefPubMedGoogle Scholar
  3. 3.
    Jungmichel S, Stucki M (2010) MDC1: the art of keeping things in focus. Chromosoma 119(4):337–349CrossRefPubMedGoogle Scholar
  4. 4.
    Jungmichel S, Clapperton JA, Lloyd J, Hari FJ, Spycher C, Pavic L, Li J, Haire LF, Bonalli M, Larsen DH, Lukas C, Lukas J, MacMillan D, Nielsen ML, Stucki M, Smerdon SJ (2012) The molecular basis of ATM-dependent dimerization of the Mdc1 DNA damage checkpoint mediator. Nucleic Acids Res 40(9):3913–3928CrossRefPubMedPubMedCentralGoogle Scholar
  5. 5.
    Bhatti S, Kozlov S, Farooqi AA, Naqi A, Lavin M, Khanna KK (2011) ATM protein kinase: the linchpin of cellular defenses to stress. Cell Mol Life Sci 68(18):2977–3006CrossRefPubMedGoogle Scholar
  6. 6.
    Kijas AW, Lim YC, 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(17):8352–8367CrossRefPubMedPubMedCentralGoogle Scholar
  7. 7.
    Lukas J, Lukas C, Bartek J (2004) Mammalian cell cycle checkpoints: signalling pathways and their organization in space and time. DNA Repair (Amst) 3(8–9):997–1007CrossRefGoogle Scholar
  8. 8.
    Lukas C, Bartek J, Lukas J (2005) Imaging of protein movement induced by chromosomal breakage: tiny ‘local’ lesions pose great 'global' challenges. Chromosoma 114(3):146–154CrossRefPubMedGoogle Scholar
  9. 9.
    Tobias F, Durante M, Taucher-Scholz G, Jakob B (2010) Spatiotemporal analysis of DNA repair using charged particle radiation. Mutat Res 704(1-3):54–60CrossRefPubMedGoogle Scholar
  10. 10.
    Tobias F, Löb D, Lengert N, Durante M, Drossel B, Taucher-Scholz G, Jakob B (2013) Spatiotemporal dynamics of early DNA damage response proteins on complex DNA lesions. PLoS One 8(2):e57953CrossRefPubMedPubMedCentralGoogle Scholar
  11. 11.
    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(36):31542–31556CrossRefPubMedPubMedCentralGoogle Scholar
  12. 12.
    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(11):9107–9119CrossRefPubMedGoogle Scholar
  13. 13.
    Splinter J, Jakob B, Lang M, Yano K, Engelhardt J, Hell SW, Chen DJ, Durante M, Taucher-Scholz G (2010) Biological dose estimation of UVA laser microirradiation utilizing charged particle-induced protein foci. Mutagenesis 25(3):289–297CrossRefPubMedPubMedCentralGoogle Scholar
  14. 14.
    Thévenaz P, Ruttimann UE, Unser M (1998) A pyramid approach to subpixel registration based on intensity. IEEE Trans Image Process 7(1):27–41CrossRefPubMedGoogle Scholar
  15. 15.
    Pataky K, Villanueva G, Liani A, Zgheib O, Jenkins N, Halazonetis DJ, Halazonetis TD, Brugger J (2009) Microcollimator for micrometer-wide stripe irradiation of cells using 20–30 keV X rays. Radiat Res 172(2):252–259CrossRefPubMedGoogle Scholar
  16. 16.
    Folkard M, Schettino G, Vojnovic B, Gilchrist S, Michette AG, Pfauntsch SJ, Prise KM, Michael BD (2001) A focused ultrasoft x-ray microbeam for targeting cells individually with submicrometer accuracy. Radiat Res 156(6):796–804CrossRefPubMedGoogle Scholar
  17. 17.
    Barberet P, Seznec H (2015) Advances in microbeam technologies and applications to radiation biology. Radiat Prot Dosimetry 166(1-4):182–187CrossRefPubMedGoogle Scholar
  18. 18.
    Jakob B, Splinter J, Conrad S, Voss KO, Zink D, Durante M, Löbrich M, Taucher-Scholz G (2011) DNA double-strand breaks in heterochromatin elicit fast repair protein recruitment, histone H2AX phosphorylation and relocation to euchromatin. Nucleic Acids Res 39(15):6489–6499CrossRefPubMedPubMedCentralGoogle Scholar
  19. 19.
    Jakob B, Splinter J, Durante M, Taucher-Scholz G (2009) Live cell microscopy analysis of radiation-induced DNA double-strand break motion. Proc Natl Acad Sci U S A 106(9):3172–3177CrossRefPubMedPubMedCentralGoogle Scholar
  20. 20.
    Stap J, Krawczyk PM, Van Oven CH, Barendsen GW, Essers J, Kanaar R, Aten JA (2008) Induction of linear tracks of DNA double-strand breaks by alpha-particle irradiation of cells. Nat Methods 5(3):261–266CrossRefPubMedGoogle Scholar
  21. 21.
    Khan R, Becker A, Taucher-Scholz G, Durante M, Fehrenbacher G, Jakob B (2014) Construction of a X-ray cabinet for live cell experiments. GSI Sci Rep 1:242Google Scholar
  22. 22.
    Abdollahi E, Taucher-Scholz G, Durante M, Jakob B (2015) Upgrading the GSI beamline microscope with a confocal fluorescence lifetime scanner to monitor charged particle induced chromatin decondensation in living cells. Nucl Instrum Methods Phys Res, Sect B 365(Pt B):626–630CrossRefGoogle Scholar
  23. 23.
    Merk B., Voss KO., Müller I.,·Fischer BE., Jakob B., Taucher-Scholz G., Trautmann C., and Durante M. (2013) Photobleaching setup for the biological end-station of the darmstadt heavy-ion microprobe. Nucl Instrum Methods Phys Res, Sect B 306, 81–84.CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media LLC 2017

Authors and Affiliations

  1. 1.GSI Helmholtzzentrum für Schwerionenforschung GmbH, BiophysikDarmstadtGermany

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