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.
Similar content being viewed by others
References
Braga J, McNally J (2007) A reaction-diffusion model to study RNA motion by quantitative fluorescence recovery after photobleaching. Biophys J 92(8):2694–2703
Burnham KP (2004) Multimodel inference: understanding AIC and BIC in model selection. Sociological Methods Res 33:261–304. doi:10.1177/0049124104268644
Burnham KP, Anderson (2002) Model selection and multi-model inference. Springer, New York
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
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
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
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
Goto M (1997) Hierarchical deterioration of body systems in Werner’s syndrome: implications for normal ageing. Mech Ageing Dev 98(3):239–254
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
Houtsmuller AB, Rademakers S, Nigg AL et al (1999) Action of DNA repair endonuclease ERCC1/XPF in living cells. Science 284:958–961
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
Indig FE, Rybanska I, Karmakar P et al (2012) Nucleolin inhibits G4 oligonucleotide unwinding by Werner helicase. PLoS One 7(6):e35229
Kass RE, Raftery AE (1995) Bayes factors. J Am Stat Assoc 90:773–795
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):6887–6892
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
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
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
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
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
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
Srivastava V, Modi P, Tripathi V, (2009) BLM helicase stimulates the ATPase and chromatin-remodeling activities of RAD54. J Cell Sci
Van Royen ME, Zotter A, Ibrahim SM, Geverts B (2011) Chromosome Res, 19:1
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
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
Acknowledgments
We would like to thank members of Center for Models of Life and the Bohr Laboratory for discussions. AT is supported by a Steno fellowship granted by the Danish Council for Independent research (DFF), KMB is supported by CMOL through the Danish National Research Foundation (DG), and MBJ is supported by the Center for Healthy Aging and an Elite Research Scholarship from the Danish Ministry of Science. This work was supported by funds from the intramural program of the National Institute of Aging, National Institutes of Health, USA.
Author information
Authors and Affiliations
Corresponding author
Additional information
K. M. Bendtsen and M. B. Jensen contributed equally.
Electronic supplementary material
Below is the link to the electronic supplementary material.
Rights and permissions
About this article
Cite this article
Bendtsen, K.M., Jensen, M.B., May, A. et al. Dynamics of the DNA repair proteins WRN and BLM in the nucleoplasm and nucleoli. Eur Biophys J 43, 509–516 (2014). https://doi.org/10.1007/s00249-014-0981-x
Received:
Revised:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00249-014-0981-x