Skip to main content
SpringerLink
Log in

Imaging of protein movement induced by chromosomal breakage: tiny ‘local’ lesions pose great ‘global’ challenges

  • Review
  • Published:
Chromosoma Aims and scope Submit manuscript

Abstract

Interruption of chromosomal integrity by DNA double-strand breaks (DSBs) causes a major threat to genomic stability. Despite tremendous progress in understanding the genetic and biochemical aspects of DSB-induced genome surveillance and repair mechanisms, little is known about organization of these molecular pathways in space and time. Here, we outline the key spatio-temporal problems associated with DSBs and focus on the imaging approaches to visualize the dynamics of DSB-induced responses in mammalian cells. We delineate benefits and limitations of these assays and highlight the key recent discoveries where live microscopy provided unprecedented insights into how cells defend themselves against genome-destabilizing effects of DNA damage.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1

Similar content being viewed by others

References

  • Aten JA, Stap J, Krawczyk PM, van Oven CH, Hoebe RA, Essers J, Kanaar R (2004) Dynamics of DNA double-strand breaks revealed by clustering of damaged chromosome domains. Science 303:92–95

    Article  PubMed  CAS  Google Scholar 

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

    Article  PubMed  CAS  Google Scholar 

  • Bakkenist CJ, Kastan MB (2004) Initiating cellular stress responses. Cell 118:9–17

    Article  PubMed  CAS  Google Scholar 

  • Bartek J, Lukas C, Lukas J (2004) Checking on DNA damage in S phase. Nat Rev Mol Cell Biol 5:792–804

    Article  PubMed  CAS  Google Scholar 

  • Bekker-Jensen S, Lukas C, Melander F, Bartek J, Lukas J (2005) Dynamic assembly and sustained retention of 53BP1 at the sites of DNA damage are coordinated by Mdc1/NFBD1. J Cell Biol, in press

  • Berns MW, Floyd AD (1971) Chromosomal microdissection by laser. A cytochemical and functional analysis. Exp Cell Res 67:305–310

    Article  PubMed  CAS  Google Scholar 

  • Berns MW, Rounds DE, Olson RS (1969) Effects of laser micro-irradiation on chromosomes. Exp Cell Res 56:292–298

    Article  PubMed  CAS  Google Scholar 

  • Berns MW, Cheng WK, Floyd AD, Onuki Y (1971) Chromosome lesions produced with an argon laser microbeam without dye sensitization. Science 171:903–905

    Article  PubMed  CAS  Google Scholar 

  • Bradshaw PS, Stavropoulos DJ, Meyn MS (2005) Human telomeric protein TRF2 associates with genomic double-strand breaks as an early response to DNA damage. Nat Genet 37:193–197

    Article  PubMed  CAS  Google Scholar 

  • Celeste A, Fernandez-Capetillo O, Kruhlak MJ, Pilch DR, Staudt DW, Lee A, Bonner RF, Bonner WM, Nussenzweig A (2003) Histone H2AX phosphorylation is dispensable for the initial recognition of DNA breaks. Nat Cell Biol 5:675–679

    Article  PubMed  CAS  Google Scholar 

  • Cremer T, Cremer C (2001) Chromosome territories, nuclear architecture and gene regulation in mammalian cells. Nat Rev Genet 2:292–310

    Article  PubMed  CAS  Google Scholar 

  • Cremer T, Cremer C, Schneider T, Baumann H, Hens L, Kirsch-Volders M (1982) Analysis of chromosome positions in the interphase nucleus of Chinese hamster cells by laser–UV–microirradiation experiments. Hum Genet 62:201–209

    Article  PubMed  CAS  Google Scholar 

  • Essers J, Houtsmuller AB, van Veelen L, Paulusma C, Nigg AL, Pastink A, Vermeulen W, Hoeijmakers JH, Kanaar R (2002) Nuclear dynamics of RAD52 group homologous recombination proteins in response to DNA damage. EMBO J 21:2030–2037

    Article  PubMed  CAS  Google Scholar 

  • Fernandez-Capetillo O, Allis CD, Nussenzweig A (2004) Phosphorylation of histone H2B at DNA double-strand breaks. J Exp Med 199:1671–1677

    Article  PubMed  CAS  Google Scholar 

  • Hauptner A, Dietzel S, Drexler GA, Reichart P, Krucken R, Cremer T, Friedl AA, Dollinger G (2004) Microirradiation of cells with energetic heavy ions. Radiat Environ Biophys 42:237–245

    Article  PubMed  CAS  Google Scholar 

  • Houtsmuller AB, Rademakers S, Nigg AL, Hoogstraten D, Hoeijmakers JH, Vermeulen W (1999) Action of DNA repair endonuclease ERCC1/XPF in living cells. Science 284:958–961

    Article  PubMed  CAS  Google Scholar 

  • Jakob B, Scholz M, Taucher-Scholz G (2002) Characterization of CDKN1A (p21) binding to sites of heavy-ion-induced damage: colocalization with proteins involved in DNA repair. Int J Radiat Biol 78:75–88

    Article  PubMed  CAS  Google Scholar 

  • Jakob B, Scholz M, Taucher-Scholz G (2003) Biological imaging of heavy charged-particle tracks. Radiat Res 159:676–684

    Article  PubMed  CAS  Google Scholar 

  • Jasin M (1996) Genetic manipulation of genomes with rare-cutting endonucleases. Trends Genet 12:224–228

    Article  PubMed  CAS  Google Scholar 

  • Jenuwein T, Allis CD (2001) Translating the histone code. Science 293:1074–1080

    Article  PubMed  CAS  Google Scholar 

  • Kastan MB, Bartek J (2004) Cell-cycle checkpoints and cancer. Nature 432:316–323

    Article  PubMed  CAS  Google Scholar 

  • Khodjakov A, Cole RW, Oakley BR, Rieder CL (2000) Centrosome-independent mitotic spindle formation in vertebrates. Curr Biol 10:59–67

    Article  PubMed  CAS  Google Scholar 

  • Kim JS, Krasieva TB, LaMorte V, Taylor AM, Yokomori K (2002a) Specific recruitment of human cohesin to laser-induced DNA damage. J Biol Chem 277:45149–45153

    Article  PubMed  CAS  Google Scholar 

  • Kim ST, Xu B, Kastan MB (2002b) Involvement of the cohesin protein, Smc1, in Atm-dependent and independent responses to DNA damage. Genes Dev 16:560–570

    Article  PubMed  CAS  Google Scholar 

  • Koundrioukoff S, Polo S, Almouzni G (2004) Interplay between chromatin and cell cycle checkpoints in the context of ATR/ATM-dependent checkpoints. DNA Repair (Amst) 3:969–978

    Article  CAS  Google Scholar 

  • Lan L, Nakajima S, Oohata Y, Takao M, Okano S, Masutani M, Wilson SH, Yasui A (2004) In situ analysis of repair processes for oxidative DNA damage in mammalian cells. Proc Natl Acad Sci U S A 101:13738–13743

    Article  PubMed  Google Scholar 

  • Limoli CL, Ward JF (1993) A new method for introducing double-strand breaks into cellular DNA. Radiat Res 134:160–169

    Article  PubMed  CAS  Google Scholar 

  • Lisby M, Mortensen UH, Rothstein R (2003) Colocalization of multiple DNA double-strand breaks at a single Rad52 repair centre. Nat Cell Biol 5:572–577

    Article  PubMed  CAS  Google Scholar 

  • Lisby M, Barlow JH, Burgess RC, Rothstein R (2004) Choreography of the DNA damage response: spatiotemporal relationships among checkpoint and repair proteins. Cell 118:699–713

    Article  PubMed  CAS  Google Scholar 

  • Lombard DB, Guarente L (2000) Nijmegen breakage syndrome disease protein and MRE11 at PML nuclear bodies and meiotic telomeres. Cancer Res 60:2331–2334

    PubMed  CAS  Google Scholar 

  • Lukas J, Bartek J (2004) Watching the DNA repair ensemble dance. Cell 118:666–668

    Article  PubMed  CAS  Google Scholar 

  • Lukas C, Falck J, Bartkova J, Bartek J, Lukas J (2003) Distinct spatiotemporal dynamics of mammalian checkpoint regulators induced by DNA damage. Nat Cell Biol 5:255–260

    Article  PubMed  CAS  Google Scholar 

  • Lukas C, Melander F, Stucki M, Falck J, Bekker-Jensen S, Goldberg M, Lerenthal Y, Jackson SP, Bartek J, Lukas J (2004a) Mdc1 couples DNA double-strand break recognition by Nbs1 with its H2AX-dependent chromatin retention. EMBO J 23:2674–2683

    Article  PubMed  CAS  Google Scholar 

  • Lukas J, Lukas C, Bartek J (2004b) Mammalian cell cycle checkpoints: signalling pathways and their organization in space and time. DNA Repair (Amst) 3:997–1007

    Article  CAS  Google Scholar 

  • Mahy NL, Perry PE, Bickmore WA (2002) Gene density and transcription influence the localization of chromatin outside of chromosome territories detectable by FISH. J Cell Biol 159:753–763

    Article  PubMed  CAS  Google Scholar 

  • Melo JA, Cohen J, Toczyski DP (2001) Two checkpoint complexes are independently recruited to sites of DNA damage in vivo. Genes Dev 15:2809–2821

    PubMed  CAS  Google Scholar 

  • Mikhailov A, Cole RW, Rieder CL (2002) DNA damage during mitosis in human cells delays the metaphase/anaphase transition via the spindle-assembly checkpoint. Curr Biol 12:1797–1806

    Article  PubMed  CAS  Google Scholar 

  • Mirzoeva OK, Petrini JH (2001) DNA damage-dependent nuclear dynamics of the Mre11 complex. Mol Cell Biol 21:281–288

    Article  PubMed  CAS  Google Scholar 

  • Mirzoeva OK, Petrini JH (2003) DNA replication-dependent nuclear dynamics of the Mre11 complex. Mol Cancer Res 1:207–218

    PubMed  CAS  Google Scholar 

  • Misteli T (2005) Concepts in nuclear architecture. BioEssays 27:477–487

    Article  PubMed  CAS  Google Scholar 

  • Nelms BE, Maser RS, MacKay JF, Lagally MG, Petrini JH (1998) In situ visualization of DNA double-strand break repair in human fibroblasts. Science 280:590–592

    Article  PubMed  CAS  Google Scholar 

  • Nyberg KA, Michelson RJ, Putnam CW, Weinert TA (2002) Toward maintaining the genome: DNA damage and replication checkpoints. Annu Rev Genet 36:617–656

    Article  PubMed  CAS  Google Scholar 

  • Osborne CS, Chakalova L, Brown KE, Carter D, Horton A, Debrand E, Goyenechea B, Mitchell JA, Lopes S, Reik W, Fraser P (2004) Active genes dynamically colocalize to shared sites of ongoing transcription. Nat Genet 36:1065–1071

    Article  PubMed  CAS  Google Scholar 

  • Paull TT, Rogakou EP, Yamazaki V, Kirchgessner CU, Gellert M, Bonner WM (2000) A critical role for histone H2AX in recruitment of repair factors to nuclear foci after DNA damage. Curr Biol 10:886–895

    Article  PubMed  CAS  Google Scholar 

  • Petrini JH, Stracker TH (2003) The cellular response to DNA double-strand breaks: defining the sensors and mediators. Trends Cell Biol 13:458–462

    Article  PubMed  CAS  Google Scholar 

  • Phair RD, Gorski SA, Misteli T (2004a) Measurement of dynamic protein binding to chromatin in vivo, using photobleaching microscopy. Methods Enzymol 375:393–414

    Article  PubMed  CAS  Google Scholar 

  • Phair RD, Scaffidi P, Elbi C, Vecerova J, Dey A, Ozato K, Brown DT, Hager G, Bustin M, Misteli T (2004b) Global nature of dynamic protein–chromatin interactions in vivo: three-dimensional genome scanning and dynamic interaction networks of chromatin proteins. Mol Cell Biol 24:6393–6402

    Article  PubMed  CAS  Google Scholar 

  • Rogakou EP, Boon C, Redon C, Bonner WM (1999) Megabase chromatin domains involved in DNA double-strand breaks in vivo. J Cell Biol 146:905–916

    Article  PubMed  CAS  Google Scholar 

  • Shiloh Y (2003) ATM and related protein kinases: safeguarding genome integrity. Nat Rev Cancer 3:155–168

    Article  PubMed  CAS  Google Scholar 

  • Shiloh Y, Lehmann AR (2004) Maintaining integrity. Nat Cell Biol 6:923–928

    Article  PubMed  CAS  Google Scholar 

  • Strom L, Lindroos HB, Shirahige K, Sjogren C (2004) Postreplicative recruitment of cohesin to double-strand breaks is required for DNA Repair. Mol Cell 16:1003–1015

    Article  PubMed  Google Scholar 

  • Tashiro S, Walter J, Shinohara A, Kamada N, Cremer T (2000) Rad51 accumulation at sites of DNA damage and in postreplicative chromatin. J Cell Biol 150:283–291

    Article  PubMed  CAS  Google Scholar 

  • Unal E, Arbel-Eden A, Sattler U, Shroff R, Lichten M, Haber JE, Koshland D (2004) DNA damage response pathway uses histone modification to assemble a double-strand break-specific cohesin domain. Mol Cell 16:991–1002

    Article  PubMed  Google Scholar 

  • Walter J, Cremer T, Miyagawa K, Tashiro S (2003) A new system for laser-UVA-microirradiation of living cells. J Microsc 209:71–75

    Article  PubMed  MathSciNet  CAS  Google Scholar 

  • Wu G, Lee WH, Chen PL (2000) NBS1 and TRF1 colocalize at promyelocytic leukemia bodies during late S/G2 phases in immortalized telomerase-negative cells. Implication of NBS1 in alternative lengthening of telomeres. J Biol Chem 275:30618–30622

    Article  PubMed  CAS  Google Scholar 

  • Wu G, Jiang X, Lee WH, Chen PL (2003) Assembly of functional ALT-associated promyelocytic leukemia bodies requires Nijmegen Breakage Syndrome 1. Cancer Res 63:2589–2595

    PubMed  CAS  Google Scholar 

  • Yang S, Kuo C, Bisi JE, Kim MK (2002) PML-dependent apoptosis after DNA damage is regulated by the checkpoint kinase hCds1/Chk2. Nat Cell Biol 4:865–870

    Article  PubMed  CAS  Google Scholar 

  • Yazdi PT, Wang Y, Zhao S, Patel N, Lee EY, Qin J (2002) SMC1 is a downstream effector in the ATM/NBS1 branch of the human S-phase checkpoint. Genes Dev 16:571–582

    Article  PubMed  CAS  Google Scholar 

  • Zhou BB, Elledge SJ (2000) The DNA damage response: putting checkpoints in perspective. Nature 408:433–439

    Article  PubMed  CAS  Google Scholar 

  • Zhu XD, Kuster B, Mann M, Petrini JH, de Lange T (2000) Cell-cycle-regulated association of RAD50/MRE11/NBS1 with TRF2 and human telomeres. Nat Genet 25:347–352

    Article  PubMed  CAS  Google Scholar 

Download references

Acknowledgements

The authors are supported by grants from the Danish Cancer Society, Danish National Research Foundation, European Union (integrated project ‘DNA repair’), European Science Foundation and John and Birthe Meyer Foundation.

Author information

Authors and Affiliations

  1. Institute of Cancer Biology and Centre for Genotoxic Stress Research, Danish Cancer Society, Strandboulevarden 49, 2100, Copenhagen, Denmark

    Claudia Lukas, Jiri Bartek & Jiri Lukas

Authors
  1. Claudia Lukas
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Jiri Bartek
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Jiri Lukas
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Jiri Lukas.

Additional information

Communicated by E.A. Nigg

Rights and permissions

Reprints and permissions

About this article

Cite this article

Lukas, C., Bartek, J. & Lukas, J. Imaging of protein movement induced by chromosomal breakage: tiny ‘local’ lesions pose great ‘global’ challenges. Chromosoma 114, 146–154 (2005). https://doi.org/10.1007/s00412-005-0011-y

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00412-005-0011-y

Keywords

Search

Navigation