UV Laser-Induced Protein-DNA Crosslinking

  • Stefan I. Dimitrov
  • Tom Moss
Part of the Methods in Molecular Biology™ book series (MIMB, volume 30)


Photochemical crosslinking is a powerful method for studying all types of protein-nucleic acids interactions. In particular, UV-induced crosslinking has been successfully applied to the study of protein-DNA interactions (1). Ultraviolet (UV) light is a zero-length crosslinking agent. It is therefore not subject to the steric problems that can be associated with chemical crosslinking agents and provides strong evidence for close protein-DNA interactions. However, to achieve an acceptable degree of crosslinking with conventional UV light sources, exposure times ranging from minutes to several hours have had to be used (1, 2, 3). Such prolonged irradiation allows for redistribution of proteins and the artifactual crosslinking of UV-damaged molecules, and it also precludes kinetic studies. The use of UV lasers overcomes these difficulties, since crosslinking is achieved after only nano- or picosecond exposures (4, 5).


Potassium Thiocyanate Prehybridization Buffer Antibody Buffer Chemical Crosslinking Agent Photochemical Crosslinking 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.


  1. 1.
    Welsh, J. and Cantor, C. R. (1984) Protein-DNA crosslinking. TIBS 9,505–507.Google Scholar
  2. 2.
    Markovitz, A. (1972) Ultraviolet light-induced stable complexes of DNA and DNA polymerase. Biochim. Biophys. Acta 281, 522–534.PubMedGoogle Scholar
  3. 3.
    Labbé, S., Préost, J, Remondelli, P., Leone, A., and Séguin, C. (1991) A nuclear factor binds to the metal regulatory elements of the mouse gene encoding metallothronein-I. Nucleic Acids Res. 19,4225–423PubMedCrossRefGoogle Scholar
  4. 4.
    Hockensmith, J. W, Kubasek, W. L., Vorachek, W R, and von Hippel, P H. (1986) Laser crosslinkmg of nucleic acids to proteins. Methodology and first apphcations to the phage T4 DNA replication system. J. Biol. Chem. 261,3512–3518.PubMedGoogle Scholar
  5. 5.
    Harrison, C. A., Turner, D. H., and Hinkle, D. C. (1982) Laser crosslinking of E. coli RNA polymerase and T7 DNA. Nucleic Acids Res. 10,2399–2414.PubMedCrossRefGoogle Scholar
  6. 6.
    Hockensmith, J. W., Kubasek, W. L., Vorachek, W. R., Evertsz, E. M., and von Hippel, P. H (1991) Meth. Enzymol. 208 211–236.PubMedCrossRefGoogle Scholar
  7. 7.
    Careri, G., Fasella, P., and Gratton, E. (1975) Stattstical time events in enzymes: a physical assessment. CRC Crit Rev. Biochem. 3, 141–164PubMedCrossRefGoogle Scholar
  8. 8.
    Pashev, I. G., Dimitrov, S. I., and Angelov, D. (1991) Crosslinking proteins to nucleic acids by ultraviolet laser-irradiation. Trends Biochem. Sci. 16,323–326.PubMedCrossRefGoogle Scholar
  9. 9.
    Angelov, D, Stefanovsky, V. Yu., Dimitrov, S. I., Russanova, V. R., Keskinova, E., and Pashev, I. G. (1988) Protein-DNA crosslinking in reconstituted nucleohtstone, nuclei and whole cells by picosecond UV laser-irradiation. Nucleic Acids Res. 16,4525–4538.PubMedCrossRefGoogle Scholar
  10. 10.
    Budowsky, E. I., Axentyeva, M. S., Abdurashidova, G. G., Simukova, N. A., and Rubin, L. B. (1986) Induction of polynucleotide-protein crosslinkages by ultraviolet irradiation. Peculiarittes of the high-intensity laser pulse irradiation. Eur. J. Biochem. 159,95–101.PubMedCrossRefGoogle Scholar
  11. 11.
    Dobrov, E. N., Arbieva, Z. K., Timofeeva, E. K., Esenahev, R. O., Oraevsky A. A., and Nikogosyan, D. N (1989) UV laser induced RNA-protein crosslinks and RNA chain breaks in tobacco mosaic virus RNA in situ. Photochem. Photobiol. 49,595–598.PubMedCrossRefGoogle Scholar
  12. 12.
    Buckle, M., Geiselmann, J, Kolb, A., and But, H. (1991) Protein-DNA crosslinking at the lac promoter. Nucleic Acids Res. 19,833–840.PubMedCrossRefGoogle Scholar
  13. 13.
    Stefanovsky, V. Y., Dimitrov, S. I., Russanova, V. R., Angelov, D., and Pashev, I. G. (1989) Laser-induced crosslinking of histones to DNA in chromatin and core particles: implications in studying histone-DNA interactions. Nucleic Acids Res 17, 1010,069–10,081.CrossRefGoogle Scholar
  14. 14.
    Stefanovsky, V. Yu., Dimitrov, S. I., Angelov, D., and Pashev, I. G. (1989) Interactions of acetylated histones with DNA as revealed by UV laser induced histone-DNA crosslinking. Biochem. Biophys. Res. Commun. 164,304–310.CrossRefGoogle Scholar
  15. 15.
    Dimitrov, S. I., Stefanovsky, V. Yu., Karagyozov, L, Angelov, D., and Pashev, I. G. (1990) The enhancers and promoters of the Xenopus laevis ribosomal spacer are associated with histones upon active transcription of the ribosomal genes. Nucleic Acids Res. l8 6393–6397.CrossRefGoogle Scholar
  16. 16.
    Feinberg, A. P. and Vogelstein, B. (1983) A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity Anal. Biochem. 132,6–13.PubMedCrossRefGoogle Scholar
  17. 17.
    Feinberg, A P. and Vogelstein, B. (1984) A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Addendum. Anal. Biochem. 137, 266,267.Google Scholar

Copyright information

© Humana Press Inc. 1994

Authors and Affiliations

  • Stefan I. Dimitrov
    • 1
  • Tom Moss
    • 1
  1. 1.Centre de Recherche en Cancérologie deI'Université LavalQuébecCanada

Personalised recommendations