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DNA Repair pp 61-74 | Cite as

In Time and Space: Laser Microirradiation and the DNA Damage Response

  • Jae Jin Kim
  • Ramhari Kumbhar
  • Fade Gong
  • Kyle M. MillerEmail author
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Part of the Methods in Molecular Biology book series (MIMB, volume 1999)

Abstract

Maintenance of genomic integrity depends on the spatiotemporal recruitment and regulation of DNA damage response and repair proteins at DNA damage sites. These highly dynamic processes have been widely studied using laser microirradiation coupled with fluorescence microscopy. Laser microirradiation has provided a powerful methodology to identify and determine mechanisms of DNA damage response pathways. Here we describe methods used to analyze protein recruitment dynamics of fluorescently tagged or endogenous proteins to laser-induced DNA damage sites using laser scanning and fluorescence microscopy. We further describe multiple applications employing these techniques to study additional processes at DNA damage sites including transcription. Together, we aim to provide robust visualization methods employing laser-microirradiation to detect and determine protein behavior, functions and dynamics in response to DNA damage in mammalian cells.

Key words

Laser microirradiation DNA repair DNA damage Transcription Fluorescence microscopy Live-cell imaging 

Notes

Acknowledgments

The K.M.M. laboratory is supported by the NIH National Cancer Institute (R01CA198279 and RO1CA201268) and the American Cancer Society (RSG-16-042-01-DMC).

References

  1. 1.
    Hoeijmakers JH (2001) Genome maintenance mechanisms for preventing cancer. Nature 411(6835):366–374.  https://doi.org/10.1038/35077232CrossRefPubMedPubMedCentralGoogle Scholar
  2. 2.
    Ciccia A, Elledge SJ (2010) The DNA damage response: making it safe to play with knives. Mol Cell 40(2):179–204.  https://doi.org/10.1016/j.molcel.2010.09.019CrossRefPubMedPubMedCentralGoogle Scholar
  3. 3.
    Jackson SP, Bartek J (2009) The DNA-damage response in human biology and disease. Nature 461(7267):1071–1078.  https://doi.org/10.1038/nature08467CrossRefPubMedPubMedCentralGoogle Scholar
  4. 4.
    Polo SE, Jackson SP (2011) Dynamics of DNA damage response proteins at DNA breaks: a focus on protein modifications. Genes Dev 25(5):409–433.  https://doi.org/10.1101/gad.2021311CrossRefPubMedPubMedCentralGoogle Scholar
  5. 5.
    Sulli G, Di Micco R, d’Adda di Fagagna F (2012) Crosstalk between chromatin state and DNA damage response in cellular senescence and cancer. Nat Rev Cancer 12(10):709–720.  https://doi.org/10.1038/nrc3344CrossRefPubMedGoogle Scholar
  6. 6.
    Aleksandrov R, Dotchev A, Poser I, Krastev D, Georgiev G, Panova G, Babukov Y, Danovski G, Dyankova T, Hubatsch L, Ivanova A, Atemin A, Nedelcheva-Veleva MN, Hasse S, Sarov M, Buchholz F, Hyman AA, Grill SW, Stoynov SS (2018) Protein dynamics in complex DNA lesions. Mol Cell 69(6):1046–1061.e1045.  https://doi.org/10.1016/j.molcel.2018.02.016CrossRefPubMedGoogle Scholar
  7. 7.
    Petrini JH, Stracker TH (2003) The cellular response to DNA double-strand breaks: defining the sensors and mediators. Trends Cell Biol 13(9):458–462CrossRefGoogle Scholar
  8. 8.
    Lee JH, Paull TT (2005) ATM activation by DNA double-strand breaks through the Mre11-Rad50-Nbs1 complex. Science 308(5721):551–554.  https://doi.org/10.1126/science.1108297CrossRefPubMedPubMedCentralGoogle Scholar
  9. 9.
    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):5858–5868CrossRefGoogle Scholar
  10. 10.
    Jackson SP, Durocher D (2013) Regulation of DNA damage responses by ubiquitin and SUMO. Mol Cell 49(5):795–807.  https://doi.org/10.1016/j.molcel.2013.01.017CrossRefPubMedGoogle Scholar
  11. 11.
    Kolas NK, Chapman JR, Nakada S, Ylanko J, Chahwan R, Sweeney FD, Panier S, Mendez M, Wildenhain J, Thomson TM, Pelletier L, Jackson SP, Durocher D (2007) Orchestration of the DNA-damage response by the RNF8 ubiquitin ligase. Science 318(5856):1637–1640.  https://doi.org/10.1126/science.1150034CrossRefPubMedPubMedCentralGoogle Scholar
  12. 12.
    Wang B, Elledge SJ (2007) Ubc13/Rnf8 ubiquitin ligases control foci formation of the Rap80/Abraxas/Brca1/Brcc36 complex in response to DNA damage. Proc Natl Acad Sci U S A 104(52):20759–20763.  https://doi.org/10.1073/pnas.0710061104CrossRefPubMedPubMedCentralGoogle Scholar
  13. 13.
    Gassman NR, Wilson SH (2015) Micro-irradiation tools to visualize base excision repair and single-strand break repair. DNA Repair (Amst) 31:52–63.  https://doi.org/10.1016/j.dnarep.2015.05.001CrossRefPubMedCentralGoogle Scholar
  14. 14.
    Mistrik M, Vesela E, Furst T, Hanzlikova H, Frydrych I, Gursky J, Majera D, Bartek J (2016) Cells and stripes: a novel quantitative photo-manipulation technique. Sci Rep 6:19567.  https://doi.org/10.1038/srep19567CrossRefPubMedPubMedCentralGoogle Scholar
  15. 15.
    Svejstrup JQ (2010) The interface between transcription and mechanisms maintaining genome integrity. Trends Biochem Sci 35(6):333–338.  https://doi.org/10.1016/j.tibs.2010.02.001CrossRefPubMedGoogle Scholar
  16. 16.
    Shanbhag NM, Rafalska-Metcalf IU, Balane-Bolivar C, Janicki SM, Greenberg RA (2010) ATM-dependent chromatin changes silence transcription in cis to DNA double-strand breaks. Cell 141(6):970–981.  https://doi.org/10.1016/j.cell.2010.04.038CrossRefPubMedPubMedCentralGoogle Scholar
  17. 17.
    Gong F, Chiu LY, Cox B, Aymard F, Clouaire T, Leung JW, Cammarata M, Perez M, Agarwal P, Brodbelt JS, Legube G, Miller KM (2015) Screen identifies bromodomain protein ZMYND8 in chromatin recognition of transcription-associated DNA damage that promotes homologous recombination. Genes Dev 29(2):197–211.  https://doi.org/10.1101/gad.252189.114CrossRefPubMedPubMedCentralGoogle Scholar
  18. 18.
    Gong F, Clouaire T, Aguirrebengoa M, Legube G, Miller KM (2017) Histone demethylase KDM5A regulates the ZMYND8-NuRD chromatin remodeler to promote DNA repair. J Cell Biol 216(7):1959–1974.  https://doi.org/10.1083/jcb.201611135CrossRefPubMedPubMedCentralGoogle Scholar
  19. 19.
    Gong F, Miller KM (2018) Double duty: ZMYND8 in the DNA damage response and cancer. Cell Cycle 17(4):414–420.  https://doi.org/10.1080/15384101.2017.1376150CrossRefPubMedPubMedCentralGoogle Scholar
  20. 20.
    Adam S, Dabin J, Chevallier O, Leroy O, Baldeyron C, Corpet A, Lomonte P, Renaud O, Almouzni G, Polo SE (2016) Real-time tracking of parental histones reveals their contribution to chromatin integrity following DNA damage. Mol Cell 64(1):65–78.  https://doi.org/10.1016/j.molcel.2016.08.019CrossRefPubMedPubMedCentralGoogle Scholar
  21. 21.
    Britton S, Coates J, Jackson SP (2013) A new method for high-resolution imaging of Ku foci to decipher mechanisms of DNA double-strand break repair. J Cell Biol 202(3):579–595.  https://doi.org/10.1083/jcb.201303073CrossRefPubMedPubMedCentralGoogle Scholar
  22. 22.
    Kong X, Cruz GMS, Silva BA, Wakida NM, Khatibzadeh N, Berns MW, Yokomori K (2018) Laser microirradiation to study in vivo cellular responses to simple and complex DNA damage. J Vis Exp (131).  https://doi.org/10.3791/56213
  23. 23.
    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):146–154.  https://doi.org/10.1007/s00412-005-0011-yCrossRefPubMedGoogle Scholar
  24. 24.
    Xie S, Mortusewicz O, Ma HT, Herr P, Poon RY, Helleday T, Qian C (2015) Timeless interacts with PARP-1 to promote homologous recombination repair. Mol Cell 60(1):163–176.  https://doi.org/10.1016/j.molcel.2015.07.031CrossRefPubMedGoogle Scholar
  25. 25.
    Dobbin MM, Madabhushi R, Pan L, Chen Y, Kim D, Gao J, Ahanonu B, Pao PC, Qiu Y, Zhao Y, Tsai LH (2013) SIRT1 collaborates with ATM and HDAC1 to maintain genomic stability in neurons. Nat Neurosci 16(8):1008–1015.  https://doi.org/10.1038/nn.3460CrossRefPubMedPubMedCentralGoogle Scholar
  26. 26.
    Wang WY, Pan L, Su SC, Quinn EJ, Sasaki M, Jimenez JC, Mackenzie IR, Huang EJ, Tsai LH (2013) Interaction of FUS and HDAC1 regulates DNA damage response and repair in neurons. Nat Neurosci 16(10):1383–1391.  https://doi.org/10.1038/nn.3514CrossRefPubMedPubMedCentralGoogle Scholar
  27. 27.
    Hustedt N, Durocher D (2016) The control of DNA repair by the cell cycle. Nat Cell Biol 19(1):1–9.  https://doi.org/10.1038/ncb3452CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  • Jae Jin Kim
    • 1
  • Ramhari Kumbhar
    • 1
  • Fade Gong
    • 1
    • 2
  • Kyle M. Miller
    • 1
    Email author
  1. 1.Department of Molecular Biosciences, Institute for Cellular and Molecular BiologyThe University of Texas at AustinAustinUSA
  2. 2.Department of Biochemistry & Molecular BiologyBaylor College of MedicineHoustonUSA

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