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Uranyl Photoprobing of DNA Structures and Drug–DNA Complexes

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Part of the Methods in Molecular Biology™ book series (MIMB,volume 90)

Abstract

The uranyl(IV) ion (UO2 2+) binds strongly to the phosphates of DNA and, upon irradiation with long wavelength ultraviolet light, the proximal deoxyn-boses are oxidized by the photochemically excited state of the uranyl1 ion, a very strong oxidant (1). Thus the uranyl ion is an efficient DNA photocleavage reagent (2,3) that has been used to study the sequence specific interaction with the phosphates of the DNA backbone of the respective DNA recogintion sites of various proteins, such as the λ-repressor/OR1 complex (3), RNA polymerase-deoP1 promoter of Escherzchza coli (4), RNA polymerase/cyclic AMP receptor protein (CRP)-deoP2 promoter of E coli (5), CRP/CytR repressor-deoP2 promoter of E. colz (5), and transcription factor IIIA/Xenopus 5s internal control region (6).

Keywords

  • Minor Groove
  • Uranyl Nitrate
  • Bromphenol Blue
  • Fluorescent Light Tube
  • Sequence Specific Interaction

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.

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  • DOI: 10.1385/0-89603-447-X:43
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References

  1. Burrows H. D. and Kemp T. J. (1974) The photochemistry of the uranyl ion. Chem Soc Rev 3, 138–165

    CrossRef  Google Scholar 

  2. Nielsen P. E., Hiort C., Buchardt O., Dahl O., Sonmchsen S. H., and Nordèn B. (1992) DNA Binding and Photocleavage by Uranyl(V1) (UO2 2+) Salts J Amer Chem. Soc 114, 4967–4975

    CAS  CrossRef  Google Scholar 

  3. Nielsen P. E., Jeppesen C., and Buchardt O. (1988) Uranyl salts as photochemical agents for cleavage of DNA and probing of protein-DNA contacts. FEBS Lett 235, 122–124

    CAS  CrossRef  Google Scholar 

  4. Jeppesen C. and Nielsen P. (1989) Uranyl mediated photofootprinting reveals strong E coli RNA polymerase-DNA backbone contacts in the + 10 region of the deoP1 promoter open complex. Nucleic Acids Res 17, 4947–4956

    CAS  CrossRef  Google Scholar 

  5. Mollegard N. E., Rasmussen P. B., Valentin-Hansen P., and Nielsen P. E. (1993) Charactertzation of promoter recognition complexes formed by CRP CytR for repression and by CRP and RNA polymerase for activation of transcription on the E. co11 deoP2 promoter J Biol Chem 268, 17,47l–17,477

    Google Scholar 

  6. Nielsen P. E. and Jeppesen C. (1990) Photochemical probing of DNA complexes. Trends Photochem Photobiol 1, 39–47

    CAS  Google Scholar 

  7. Nielsen P. E., Mollegaard N. E. and Jeppesen C. (1990) DNA conformational analysis in solution by uranyl mediated photocleavage Nucleic Acids Res 18, 3847–3851

    CAS  CrossRef  Google Scholar 

  8. Mollegaard N. E. (1992) Uranyl photoprobing of DNA structures and protein DNA interactions, Thesis

    Google Scholar 

  9. Sonnichsen S. H. and Nielsen P. E. (1996) J Mol Recognition 9, 219–227

    CAS  CrossRef  Google Scholar 

  10. Mollegaard N. E., Murchie A. I., Lilley D. M., and Nielsen P. E. (1994) Uranyl photoprobing of a four-way DNA Junction. Evidence for spectfic metal ion binding EMBO J 13, 1508–1513

    CAS  Google Scholar 

  11. Nielsen P. E. and Mollegaard N. E. (1996) J Mel Recognition 9, 228–232

    CAS  CrossRef  Google Scholar 

  12. Sambrook J., Fritsch E. F., and Maniatis T. (1982) Molecular Cloning A Laboratory Manual Cold Spring Harbor Laboratory, Cold Spring Harbor, NY

    Google Scholar 

  13. Portugal J. and Waring M. J. (1987) Eur J Biochem. 167, 28l–289

    CrossRef  Google Scholar 

  14. Portugal J. and Waring M. J. (1987). Hydroxyl radical footprinting of the sequence-selective binding of netropsin and distamycin FEBS Lett. 225, 195–200.

    CAS  CrossRef  Google Scholar 

  15. Coll M., Frederick C. A., Wang A. H., and Rich A. (1987) Proc Natl Acad Sci USA 84, 8385–8389

    CAS  CrossRef  Google Scholar 

  16. Wood A. A., Nunn C. M., Boykin D. W., and Needle S. (1995) Nucleic Acids Res 23, 3678–3684.

    CAS  CrossRef  Google Scholar 

  17. Nielsen P. E., Cons B. M. G., Fox K. R., and Sommer V. B. (1990) Uranyl photofootprinting DNA structural changes upon binding of mithramycin, in Molecular Basis of Specificity in Nucleic Acid Drug Interactions, vol. 23(Pullman B and Jortner J, eds), The Jerusalem Symposium on Quantum Chemistry and Biochemistry, Dordrecht, pp. 423–432

    CrossRef  Google Scholar 

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© 1997 Humana Press Inc., Totowa NJ

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Møllegaard, N.E., Nielsen, P.E. (1997). Uranyl Photoprobing of DNA Structures and Drug–DNA Complexes. In: Fox, K.R. (eds) Drug-DNA Interaction Protocols. Methods in Molecular Biology™, vol 90. Humana Press, Totowa, NJ. https://doi.org/10.1385/0-89603-447-X:43

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  • DOI: https://doi.org/10.1385/0-89603-447-X:43

  • Publisher Name: Humana Press, Totowa, NJ

  • Print ISBN: 978-0-89603-447-1

  • Online ISBN: 978-1-59259-574-7

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