AIMS Genetics, 2017, 4(1): 47-68. doi: 10.3934/genet.2017.1.47.

Research article

Export file:


  • RIS(for EndNote,Reference Manager,ProCite)
  • BibTex
  • Text


  • Citation Only
  • Citation and Abstract

Systematic analysis of DNA damage induction and DNA repair pathway activation by continuous wave visible light laser micro-irradiation

Cell Biology and Epigenetics, Department of Biology, Technische Universität Darmstadt, 64287 Darmstadt, Germany

Laser micro-irradiation can be used to induce DNA damage with high spatial and temporal resolution, representing a powerful tool to analyze DNA repair in vivo in the context of chromatin. However, most lasers induce a mixture of DNA damage leading to the activation of multiple DNA repair pathways and making it impossible to study individual repair processes. Hence, we aimed to establish and validate micro-irradiation conditions together with inhibition of several key proteins to discriminate different types of DNA damage and repair pathways using lasers commonly available in confocal microscopes. Using time-lapse analysis of cells expressing fluorescently tagged repair proteins and also validation of the DNA damage generated by micro-irradiation using several key damage markers, we show that irradiation with a 405 nm continuous wave laser lead to the activation of all repair pathways even in the absence of exogenous sensitization. In contrast, we found that irradiation with 488 nm laser lead to the selective activation of non-processive short-patch base excision and single strand break repair, which were further validated by PARP inhibition and metoxyamine treatment. We conclude that these low energy conditions discriminated against processive long-patch base excision repair, nucleotide excision repair as well as double strand break repair pathways.
  Article Metrics

Keywords DNA repair; DNA damage; processive DNA synthesis; laser micro-irradiation; live-cell microscopy

Citation: Britta Muster, Alexander Rapp, M. Cristina Cardoso. Systematic analysis of DNA damage induction and DNA repair pathway activation by continuous wave visible light laser micro-irradiation. AIMS Genetics, 2017, 4(1): 47-68. doi: 10.3934/genet.2017.1.47


  • 1. Hegde ML, Hazra TK, Mitra S (2008) Early steps in the DNA base excision/single-strand interruption repair pathway in mammalian cells. Cell Res 18: 27-47.    
  • 2. Hoeijmakers JH (2009) DNA damage, aging, and cancer. N Engl J Med 361: 1475-1485.    
  • 3. Goodarzi AA, Jeggo PA (2013) The repair and signaling responses to DNA double-strand breaks. Adv Genet 82: 1-45.
  • 4. Polo SE, Jackson SP (2011) Dynamics of DNA damage response proteins at DNA breaks: a focus on protein modifications. Genes Dev 25: 409-433.    
  • 5. Mortusewicz O, Leonhardt H, Cardoso MC (2008) Spatiotemporal dynamics of regulatory protein recruitment at DNA damage sites. J CELL Biochem 104: 1562-1569.    
  • 6. Gassman NR, Wilson SH (2015) Micro-irradiation tools to visualize base excision repair and single-strand break repair. DNA Repair 31: 52-63.    
  • 7. Lengert L, Lengert N, Drossel B, et al. (2015) Discrimination of Kinetic Models by a Combination of Microirradiation and Fluorescence Photobleaching. Biophys J 109: 1551-1564.    
  • 8. Bekker-Jensen S, Lukas C, Kitagawa R, et al. (2006) Spatial organization of the mammalian genome surveillance machinery in response to DNA strand breaks. J Cell Biol 173: 195-206.    
  • 9. Cremer C, Cremer T, Fukuda M, et al. (1980) Detection of laser--UV microirradiation-induced DNA photolesions by immunofluorescent staining. Hum Genet 54: 107-110.    
  • 10. Lukas C, Falck J, Bartkova J, et al. (2003) Distinct spatiotemporal dynamics of mammalian checkpoint regulators induced by DNA damage. Nat Cell Biol 5: 255-260.    
  • 11. Lukas C, Melander F, Stucki M, et al. (2004) Mdc1 couples DNA double-strand break recognition by Nbs1 with its H2AX-dependent chromatin retention. EMBO J 23: 2674-2683.    
  • 12. Rogakou EP, Boon C, Redon C, et al. (1999) Megabase chromatin domains involved in DNA double-strand breaks in vivo. J Cell Biol 146: 905-916.    
  • 13. Tashiro S, Walter J, Shinohara A, et al. (2000) Rad51 accumulation at sites of DNA damage and in postreplicative chromatin. J Cell Biol 150: 283-291.    
  • 14. Kong X, Mohanty SK, Stephens J, et al. (2009) Comparative analysis of different laser systems to study cellular responses to DNA damage in mammalian cells. Nucleic Acids Res 37: e68.    
  • 15. Dinant C, de Jager M, Essers J, et al. (2007) Activation of multiple DNA repair pathways by sub-nuclear damage induction methods. J Cell Sci 120: 2731-2740.    
  • 16. Lan L, Nakajima S, Oohata Y, et al. (2004) In situ analysis of repair processes for oxidative DNA damage in mammalian cells. Proc Natl Acad Sci U S A 101: 13738-13743.    
  • 17. Mortusewicz O, Leonhardt H (2007) XRCC1 and PCNA are loading platforms with distinct kinetic properties and different capacities to respond to multiple DNA lesions. BMC molecular biology 8: 81.    
  • 18. Mortusewicz O, Rothbauer U, Cardoso MC, et al. (2006) Differential recruitment of DNA Ligase I and III to DNA repair sites. Nucleic Acids Res 34: 3523-3532.    
  • 19. Mortusewicz O, Schermelleh L, Walter J, et al. (2005) Recruitment of DNA methyltransferase I to DNA repair sites. Proc Natl Acad Sci U S A 102: 8905-8909.    
  • 20. Campalans A, Kortulewski T, Amouroux R, et al. (2013) Distinct spatiotemporal patterns and PARP dependence of XRCC1 recruitment to single-strand break and base excision repair. Nucleic Acids Res 41: 3115-3129.    
  • 21. Menoni H, Hoeijmakers JH, Vermeulen W (2012) Nucleotide excision repair-initiating proteins bind to oxidative DNA lesions in vivo. J Cell Biol 199: 1037-1046.    
  • 22. Reynolds P, Botchway SW, Parker AW, et al. (2013) Spatiotemporal dynamics of DNA repair proteins following laser microbeam induced DNA damage - when is a DSB not a DSB? Mutat Res 756: 14-20.    
  • 23. Trautlein D, Deibler M, Leitenstorfer A, et al. (2010) Specific local induction of DNA strand breaks by infrared multi-photon absorption. Nucleic Acids Res 38: e14.    
  • 24. Fischer JM, Popp O, Gebhard D, et al. (2014) Poly(ADP-ribose)-mediated interplay of XPA and PARP1 leads to reciprocal regulation of protein function. FEBS J 281: 3625-3641.    
  • 25. Reynolds P, Anderson JA, Harper JV, et al. (2012) The dynamics of Ku70/80 and DNA-PKcs at DSBs induced by ionizing radiation is dependent on the complexity of damage. Nucleic Acids Res 40: 10821-10831.    
  • 26. Ferrando-May E, Tomas M, Blumhardt P, et al. (2013) Highlighting the DNA damage response with ultrashort laser pulses in the near infrared and kinetic modeling. Front Genet 4: 135.
  • 27. Mohanty SK, Rapp A, Monajembashi S, et al. (2002) Comet assay measurements of DNA damage in cells by laser microbeams and trapping beams with wavelengths spanning a range of 308 nm to 1064 nm. Radiat Res 157: 378-385.
  • 28. Kim JS, Heale JT, Zeng W, et al. (2007) In situ analysis of DNA damage response and repair using laser microirradiation. Methods Cell Biol 82: 377-407.    
  • 29. Landry JJ, Pyl PT, Rausch T, et al. (2013) The genomic and transcriptomic landscape of a HeLa cell line. G3 (Bethesda) 3: 1213-1224.    
  • 30. Trucco C, Oliver FJ, de Murcia G, et al. (1998) DNA repair defect in poly(ADP-ribose) polymerase-deficient cell lines. Nucleic Acids Res 26: 2644-2649.    
  • 31. Oshima RG, Pellett OL, Robb JA, et al. (1977) Transformation of human cystinotic fibroblasts by SV40: characteristics of transformed cells with limited and unlimited growth potential. J Cell Physiol 93: 129-136.    
  • 32. Chagin VO, Casas-Delucchi CS, Reinhart M, et al. (2016) 4D Visualization of replication foci in mammalian cells corresponding to individual replicons. Nat Commun 7: 11231.    
  • 33. Casas-Delucchi CS, Becker A, Bolius JJ, et al. (2012) Targeted manipulation of heterochromatin rescues MeCP2 Rett mutants and re-establishes higher order chromatin organization. Nucleic Acids Res 40: e176.    
  • 34. Mortusewicz O, Fouquerel E, Ame JC, et al. (2011) PARG is recruited to DNA damage sites through poly(ADP-ribose)- and PCNA-dependent mechanisms. Nucleic Acids Res 39: 5045-5056.    
  • 35. Sporbert A, Domaing P, Leonhardt H, et al. (2005) PCNA acts as a stationary loading platform for transiently interacting Okazaki fragment maturation proteins. Nucleic Acids Res 33: 3521-3528.    
  • 36. Rodgers W, Jordan SJ, Capra JD (2002) Transient association of Ku with nuclear substrates characterized using fluorescence photobleaching. J Immunol 168: 2348-2355.    
  • 37. Bergink S, Toussaint W, Luijsterburg MS, et al. (2012) Recognition of DNA damage by XPC coincides with disruption of the XPC-RAD23 complex. J Cell Biol 196: 681-688.    
  • 38. Hoogstraten D, Bergink S, Ng JM, et al. (2008) Versatile DNA damage detection by the global genome nucleotide excision repair protein XPC. J Cell Sci 121: 2850-2859.    
  • 39. Pohler JR, Otterlei M, Warbrick E (2005) An in vivo analysis of the localisation and interactions of human p66 DNA polymerase delta subunit. BMC molecular biology 6: 17.    
  • 40. Grigaravicius P, Greulich KO, Monajembashi S (2009) Laser microbeams and optical tweezers in ageing research. ChemPhysChem 10: 79-85.    
  • 41. Henricksen LA, Umbricht CB, Wold MS (1994) Recombinant replication protein A: expression, complex formation, and functional characterization. J Biol Chem 269: 11121-11132.
  • 42. Tang JB, Goellner EM, Wang XH, et al. (2010) Bioenergetic metabolites regulate base excision repair-dependent cell death in response to DNA damage. Mol Cancer Res 8: 67-79.    
  • 43. Sakaue-Sawano A, Kurokawa H, Morimura T, et al. (2008) Visualizing spatiotemporal dynamics of multicellular cell-cycle progression. Cell 132: 487-498.    
  • 44. Lan L, Nakajima S, Komatsu K, et al. (2005) Accumulation of Werner protein at DNA double-strand breaks in human cells. J Cell Sci 118: 4153-4162.    
  • 45. Splinter J, Jakob B, Lang M, et al. (2010) Biological dose estimation of UVA laser microirradiation utilizing charged particle-induced protein foci. Mutagenesis 25: 289-297.    
  • 46. Solarczyk KJ, Zarebski M, Dobrucki JW (2012) Inducing local DNA damage by visible light to study chromatin repair. DNA Repair 11: 996-1002.    
  • 47. Sutherland JC, Griffin KP (1981) Absorption spectrum of DNA for wavelengths greater than 300 nm. Radiat Res 86: 399-409.    
  • 48. Greinert R, Volkmer B, Henning S, et al. (2012) UVA-induced DNA double-strand breaks result from the repair of clustered oxidative DNA damages. Nucleic Acids Res 40: 10263-10273.    
  • 49. Kielbassa C, Roza L, Epe B (1997) Wavelength dependence of oxidative DNA damage induced by UV and visible light. Carcinogenesis 18: 811-816.    
  • 50. Pflaum M, Boiteux S, Epe B (1994) Visible light generates oxidative DNA base modifications in high excess of strand breaks in mammalian cells. Carcinogenesis 15: 297-300.    
  • 51. Povirk LF, Wubter W, Kohnlein W, et al. (1977) DNA double-strand breaks and alkali-labile bonds produced by bleomycin. Nucleic Acids Res 4: 3573-3580.    
  • 52. Raderschall E, Bazarov A, Cao J, et al. (2002) Formation of higher-order nuclear Rad51 structures is functionally linked to p21 expression and protection from DNA damage-induced apoptosis. J Cell Sci 115: 153-164.
  • 53. Rapp A, Greulich KO (2004) After double-strand break induction by UV-A, homologous recombination and nonhomologous end joining cooperate at the same DSB if both systems are available. J Cell Sci 117: 4935-4945.    
  • 54. Trujillo KM, Yuan SS, Lee EY, et al. (1998) Nuclease activities in a complex of human recombination and DNA repair factors Rad50, Mre11, and p95. J Biol Chem 273: 21447-21450.    
  • 55. Fernandez-Capetillo O, Celeste A, Nussenzweig A (2003) Focusing on foci: H2AX and the recruitment of DNA-damage response factors. Cell Cycle 2: 426-427.
  • 56. Cadet J, Mouret S, Ravanat JL, et al. (2012) Photoinduced damage to cellular DNA: direct and photosensitized reactions. Photochem Photobiol 88: 1048-1065.    
  • 57. Sugasawa K, Okamoto T, Shimizu Y, et al. (2001) A multistep damage recognition mechanism for global genomic nucleotide excision repair. Genes Dev 15: 507-521.    
  • 58. Caldecott KW (2003) XRCC1 and DNA strand break repair. DNA Repair 2: 955-969.    
  • 59. Reynolds P, Cooper S, Lomax M, et al. (2015) Disruption of PARP1 function inhibits base excision repair of a sub-set of DNA lesions. Nucleic Acids Res 43: 4028-4038.    
  • 60. Strom CE, Johansson F, Uhlen M, et al. (2011) Poly (ADP-ribose) polymerase (PARP) is not involved in base excision repair but PARP inhibition traps a single-strand intermediate. Nucleic Acids Res 39: 3166-3175.    
  • 61. Caldecott KW (2008) Single-strand break repair and genetic disease. Nat Rev Genet 9: 619-631.
  • 62. Caldecott KW (2014) DNA single-strand break repair. Exp Cell Res 329: 2-8.    
  • 63. Prasad R, Shock DD, Beard WA, et al. (2010) Substrate channeling in mammalian base excision repair pathways: passing the baton. J Biol Chem 285: 40479-40488.    
  • 64. Underhill C, Toulmonde M, Bonnefoi H (2011) A review of PARP inhibitors: from bench to bedside. Ann Oncol 22: 268-279.    
  • 65. Kleppa L, Mari PO, Larsen E, et al. (2012) Kinetics of endogenous mouse FEN1 in base excision repair. Nucleic Acids Res 40: 9044-9059.    
  • 66. Liu L, Gerson SL (2004) Therapeutic impact of methoxyamine: blocking repair of abasic sites in the base excision repair pathway. Curr Opin Investig Drugs 5: 623-627.
  • 67. Vidal AE, Boiteux S, Hickson ID, et al. (2001) XRCC1 coordinates the initial and late stages of DNA abasic site repair through protein-protein interactions. EMBO J 20: 6530-6539.    


This article has been cited by

  • 1. Jakub A. Kochan, Emilie C.B. Desclos, Ruben Bosch, Luna Meister, Lianne E.M. Vriend, Haico v. Attikum, Przemek M. Krawczyk, Meta-analysis of DNA double-strand break response kinetics, Nucleic Acids Research, 2017, 10.1093/nar/gkx1128
  • 2. Alfonso Blázquez-Castro, Optical Tweezers: Phototoxicity and Thermal Stress in Cells and Biomolecules, Micromachines, 2019, 10, 8, 507, 10.3390/mi10080507
  • 3. Giulio Ticli, Ennio Prosperi, In Situ Analysis of DNA-Protein Complex Formation upon Radiation-Induced DNA Damage, International Journal of Molecular Sciences, 2019, 20, 22, 5736, 10.3390/ijms20225736
  • 4. Vadim O. Chagin, Britta Reinhart, Annette Becker, Oliver Mortusewicz, K. Laurence Jost, Alexander Rapp, Heinrich Leonhardt, M. Cristina Cardoso, Processive DNA synthesis is associated with localized decompaction of constitutive heterochromatin at the sites of DNA replication and repair, Nucleus, 2019, 10, 1, 231, 10.1080/19491034.2019.1688932

Reader Comments

your name: *   your email: *  

Copyright Info: 2017, M. Cristina Cardoso, et al., licensee AIMS Press. This is an open access article distributed under the terms of the Creative Commons Attribution Licese (

Download full text in PDF

Export Citation

Copyright © AIMS Press All Rights Reserved