European Biophysics Journal

, Volume 43, Issue 10–11, pp 509–516 | Cite as

Dynamics of the DNA repair proteins WRN and BLM in the nucleoplasm and nucleoli

  • Kristian Moss Bendtsen
  • Martin Borch Jensen
  • Alfred May
  • Lene Juel Rasmussen
  • Ala Trusina
  • Vilhelm A. Bohr
  • Mogens H. Jensen
Original Paper

Abstract

We have investigated the mobility of two EGFP-tagged DNA repair proteins, WRN and BLM. In particular, we focused on the dynamics in two locations, the nucleoli and the nucleoplasm. We found that both WRN and BLM use a “DNA-scanning” mechanism, with rapid binding–unbinding to DNA resulting in effective diffusion. In the nucleoplasm WRN and BLM have effective diffusion coefficients of 1.62 and 1.34 μm2/s, respectively. Likewise, the dynamics in the nucleoli are also best described by effective diffusion, but with diffusion coefficients a factor of ten lower than in the nucleoplasm. From this large reduction in diffusion coefficient we were able to classify WRN and BLM as DNA damage scanners. In addition to WRN and BLM we also classified other DNA damage proteins and found they all fall into one of two categories. Either they are scanners, similar to WRN and BLM, with very low diffusion coefficients, suggesting a scanning mechanism, or they are almost freely diffusing, suggesting that they interact with DNA only after initiation of a DNA damage response.

Keywords

DNA damage FRAP Diffusion DNA repair WRN BLM 

Supplementary material

249_2014_981_MOESM1_ESM.docx (1.8 mb)
Supplementary material 1 (DOCX 1847 kb)

References

  1. Braga J, McNally J (2007) A reaction-diffusion model to study RNA motion by quantitative fluorescence recovery after photobleaching. Biophys J 92(8):2694–2703PubMedCrossRefPubMedCentralGoogle Scholar
  2. Burnham KP (2004) Multimodel inference: understanding AIC and BIC in model selection. Sociological Methods Res 33:261–304. doi:10.1177/0049124104268644 CrossRefGoogle Scholar
  3. Burnham KP, Anderson (2002) Model selection and multi-model inference. Springer, New YorkGoogle Scholar
  4. Compton SA, Tolun G, Kamath-Loeb AS et al (2008) The Werner syndrome protein binds replication fork and holliday junction DNAs as an oligomer. J Biol Chem 283:24478–24483. doi:10.1074/jbc.M803370200 PubMedCrossRefPubMedCentralGoogle Scholar
  5. Eladad S (2005) Intra-nuclear trafficking of the BLM helicase to DNA damage-induced foci is regulated by SUMO modification. Hum Mol Genet 14:1351–1365. doi:10.1093/hmg/ddi145 PubMedCrossRefGoogle Scholar
  6. Erickson HP (2009) Size and shape of protein molecules at the nanometer level determined by sedimentation, gel filtration, and electron microscopy. Biol Proced Online 11:32–51. doi:10.1007/s12575-009-9008-x PubMedCrossRefPubMedCentralGoogle Scholar
  7. Futami K, Ishikawa Y, Goto M et al (2008) Role of Werner syndrome gene product helicase in carcinogenesis and in resistance to genotoxins by cancer cells. Cancer Sci 99:843–848. doi:10.1111/j.1349-7006.2008.00778.x PubMedCrossRefGoogle Scholar
  8. Goto M (1997) Hierarchical deterioration of body systems in Werner’s syndrome: implications for normal ageing. Mech Ageing Dev 98(3):239–254PubMedCrossRefGoogle Scholar
  9. Grierson PM, Lillard K, Behbehani GK et al (2012) BLM helicase facilitates RNA polymerase I-mediated ribosomal RNA transcription. Hum Mol Genet 21:1172–1183. doi:10.1093/hmg/ddr545 PubMedCrossRefPubMedCentralGoogle Scholar
  10. Houtsmuller AB, Rademakers S, Nigg AL et al (1999) Action of DNA repair endonuclease ERCC1/XPF in living cells. Science 284:958–961PubMedCrossRefGoogle Scholar
  11. Huranová M, Ivani I, Benda A et al (2010) The differential interaction of snRNPs with pre-mRNA reveals splicing kinetics in living cells. J Cell Biol 191:75–86. doi:10.1083/jcb.201004030 PubMedCrossRefPubMedCentralGoogle Scholar
  12. Indig FE, Rybanska I, Karmakar P et al (2012) Nucleolin inhibits G4 oligonucleotide unwinding by Werner helicase. PLoS One 7(6):e35229PubMedCrossRefPubMedCentralGoogle Scholar
  13. Kass RE, Raftery AE (1995) Bayes factors. J Am Stat Assoc 90:773–795CrossRefGoogle Scholar
  14. Marciniak RA, Lombard DB, Johnson FB, Guarente L (1998) Nucleolar localization of the Werner syndrome protein in human cells. Proc Natl Acad Sci USA 95(12):6887–6892PubMedCrossRefPubMedCentralGoogle Scholar
  15. Misteli T, Soutoglou E (2009) The emerging role of nuclear architecture in DNA repair and genome maintenance. Nat Rev Mol Cell Biol 10:243–254. doi:10.1038/nrm2651 PubMedCrossRefPubMedCentralGoogle Scholar
  16. Mueller F, Wach P, McNally JG (2008) Evidence for a common mode of transcription factor interaction with chromatin as revealed by improved quantitative fluorescence recovery after photobleaching. Biophys J 94:3323–3339. doi:10.1529/biophysj.107.123182 PubMedCrossRefPubMedCentralGoogle Scholar
  17. Rossi ML, Ghosh AK, Bohr VA (2010) Roles of Werner syndrome protein in protection of genome integrity. DNA Repair 9:331–344. doi:10.1016/j.dnarep.2009.12.011 PubMedCrossRefPubMedCentralGoogle Scholar
  18. Shiratori M, Suzuki T, Itoh C et al (2002) WRN helicase accelerates the transcription of ribosomal RNA as a component of an RNA polymerase I-associated complex. Nature Oncogene 1–8:2447–2454. doi:10.1038/sj CrossRefGoogle Scholar
  19. Spiess A-N, Neumeyer N (2010) An evaluation of R2 as an inadequate measure for nonlinear models in pharmacological and biochemical research: a Monte Carlo approach. BMC Pharmacol 10:6. doi:10.1186/1471-2210-10-6 PubMedCrossRefPubMedCentralGoogle Scholar
  20. Sprague BL, Pego RL, Stavreva DA, McNally JG (2004) Analysis of binding reactions by fluorescence recovery after photobleaching. Biophys J 86:3473–3495. doi:10.1529/biophysj.103.026765 PubMedCrossRefPubMedCentralGoogle Scholar
  21. Srivastava V, Modi P, Tripathi V, (2009) BLM helicase stimulates the ATPase and chromatin-remodeling activities of RAD54. J Cell SciGoogle Scholar
  22. Van Royen ME, Zotter A, Ibrahim SM, Geverts B (2011) Chromosome Res, 19:1Google Scholar
  23. von Kobbe C (2002) Colocalization, physical, and functional interaction between Werner and Bloom syndrome proteins. J Biol Chem 277:22035–22044. doi:10.1074/jbc.M200914200 CrossRefGoogle Scholar
  24. Yankiwski V, Marciniak RA, Guarente L, Neff NF (2000) Nuclear structure in normal and bloom syndrome cells. Proc Natl Acad Sci USA 97:5214–5219. doi:10.1073/pnas.090525897 PubMedCrossRefPubMedCentralGoogle Scholar

Copyright information

© European Biophysical Societies' Association 2014

Authors and Affiliations

  • Kristian Moss Bendtsen
    • 1
  • Martin Borch Jensen
    • 2
    • 4
  • Alfred May
    • 3
  • Lene Juel Rasmussen
    • 2
  • Ala Trusina
    • 1
  • Vilhelm A. Bohr
    • 2
    • 3
  • Mogens H. Jensen
    • 1
  1. 1.CMOL, Niels Bohr InstituteUniversity of CopenhagenCopenhagenDenmark
  2. 2.Center for Healthy Aging, Department of Cellular and Molecular MedicineUniversity of CopenhagenCopenhagenDenmark
  3. 3.National Institute on AgingBaltimoreUSA
  4. 4.Buck Institute for Research on AgingNovatoUSA

Personalised recommendations