Skip to main content
SpringerLink
Log in

The Use of Diethyl Pyrocarbonate and Potassium Permanganate as Probes for Strand Separation and Structural Distortions in DNA

  • Protocol
  • First Online:
DNA-Protein Interactions

Part of the book series: Methods in Molecular Biology™ ((MIMB,volume 543))

Summary

Diethyl pyrocarbonate (DEPC) and potassium permanganate are useful reagents for detecting DNA distortions, especially melted regions. Unlike most other footprinting methods, these reagents can detect such distortions even within the regions of protein–DNA complexes normally protected in other footprinting techniques. Further, reactions are very robust, so that distorted regions can be detected even under conditions where efficiency of DNA–protein complex formation is not high. DEPC reacts with bases that are fully or partially unstacked in DNA, in the preferential order adenosine > guanine >> cytosine. Permanganate reacts strongly with thymine in unstacked regions of DNA, and exhibits only very weak reaction with guanine, cytosine, or adenine. The combination of both reagents gives excellent coverage of all sequence regions of DNA. Because reaction requires unstacking, the two reagents detect both melted regions and regions that are unstacked because of other distortions such as bending. Permanganate has the additional advantage that it can be utilized in living cells.

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

Access this chapter

Log in via an institution
Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 129.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 169.00
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 169.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  1. Leonard, N.J., McDonald, J.J., and Reichmann, M.E. (1970). Reaction of diethyl pyrocarbonate with nucleic acid components I: Adenine. Proc. Natl Acad. Sci. USA 67, 93–98.

    Article  PubMed  CAS  Google Scholar 

  2. Leonard, N.J., McDonald, J.J., Henderson, R.E.L., and Reichmann, M.E. (1971). Reaction of diethyl pyrocarbonate with nucleic acid components: Adenosine. Biochemistry 10, 3335–3342.

    Article  PubMed  CAS  Google Scholar 

  3. Mendel, D., and Dervan, P.B. (1987). Hoogsteen base pairs proximal and distal to echinomycin binding sites on DNA. Proc. Natl Acad. Sci. USA 84, 910–914.

    Article  PubMed  CAS  Google Scholar 

  4. Hayatsu, H., and Ukita, T. (1967). The selective degredation of pyrimidines in nucleic acids by permanganate oxidation. Biochem. Biophys. Res. Commun 29, 556–561.

    Article  PubMed  CAS  Google Scholar 

  5. Howgate, P., Jones, A.S., and Tittensor, J.J. (1968). The permanganate oxidation of thymidine. J. Chem. Soc. C 1, 275–279.

    Article  Google Scholar 

  6. Iida, S., and Hayatsu, H. (1971). The permanganate oxidation of thymidine. J. Biophys. Acta 240, 370–375.

    CAS  Google Scholar 

  7. Rubin, C.M., and Schmid, C.W. (1980). Pyrimidine-specific chemical reactions useful for DNA sequencing. Nucleic Acids Res. 8, 4613–4619.

    Article  PubMed  CAS  Google Scholar 

  8. Akman, S.A., Doroshow, J.H., and Dizdaroglu, M. (1990). Base modifications in plasmid DNA caused by potassium permanganate. Arch. Biochem. Biophys 282, 202–205.

    Article  PubMed  CAS  Google Scholar 

  9. Akman, S.A., Forrest, G.P., Doroshaw, J.H., and Dizdaroglu, M. (1991). Mutation of potassium permanganate- and hydrogen peroxide-treated plasmid pZ189 replicating in CV-1 monkey kidney cells. Mutat. Res 261, 123–130.

    Article  PubMed  CAS  Google Scholar 

  10. Sasse-Dwight, S., and Gralla, D. (1988). Probing the Escherichia coli glnALG upstream activation mechanism in vivo. Proc. Natl Acad. Sci. USA 85, 8934–8938.

    Article  PubMed  CAS  Google Scholar 

  11. Sasse-Dwight, S. and Gralla, D. (1989). KMnO4 as a probe for lac promoter DNA melting and mechanism in vivo. Proc. Natl Acad. Sci. USA 264, 8074–8081.

    CAS  Google Scholar 

  12. Grimes, E., Busby, S., and Minchin, S. (1991). Differential thermal energy requirement for open complex formation by Escherichia coli RNA polymerase at two related promoters. Nucleic Acids Res. 19, 6113–6118.

    Article  PubMed  CAS  Google Scholar 

  13. Suh, W.C., Ross, W., and Record, M.T., Jr. (1992). Two open complexes and a requirement for magnesium to open the lambda-P-R transcription start site. Science 259, 358–361.

    Article  Google Scholar 

  14. Lofquist, A.K., Li, H., Imboden, M.A., and Paule, M.R. (1993). Promoter opening (melting) and transcription initiation by RNA polymerase I requires neither nucleotide β,γ hydrolysis nor protein phosphorylation. Nucleic Acids Res. 21, 3233–3238.

    Article  PubMed  CAS  Google Scholar 

  15. Kassavetis, G.A., Blanco, J.A., Johnson, T.E., and Geiduschek, E.P. (1992). Formation of open and elongating transcription complexes by RNA polymerase III. J. Mol. Biol. 226, 47–58.

    Article  PubMed  CAS  Google Scholar 

  16. Wong, C., and Gralla, J.D. (1992). A role for the acidic repeat region of transcription factor sigma 54 in setting the rate and temperature dependence of promoter melting in vivo. J. Biol. Chem. 267, 24762–24768.

    PubMed  CAS  Google Scholar 

  17. Kainz, M., and Roberts, J. (1992). Structures of transcription elongation complexes in vivo. Science 255, 838–841.

    Article  PubMed  CAS  Google Scholar 

  18. Ohlsen, K.L., and Gralla, J.D. (1992). Melting during steady-state transcription of the RRNB P-1 promoter in vivo and in vitro. J. Bacteriol. 174, 6071–6075.

    PubMed  CAS  Google Scholar 

  19. Li, B., Weber, J.A., Chen, Y., Greenleaf, A.L., and Gilmour, D.S. (1996). Analysis of promoter-proximal pausing by RNA polymerase II on the hsp70 heat shock gene promoter in a Drosophila nuclear extract. Mol. Cell. Biol. 16, 5433–5443.

    PubMed  CAS  Google Scholar 

  20. Hartvig, L., and Christiansen, J. (1996). Intrinsic termination of T7 RNA polymerase mediated by either RNA or DNA. EMBO J. 15, 4767–4774.

    PubMed  CAS  Google Scholar 

  21. Komissarova, N., and Kashlev, M. (1997). Transcriptional arrest: Escherichia coli RNA polymerase translocates backward, leaving the 3′ end of the RNA intact and extruded. Proc. Natl Acad. Sci. USA 94, 1755–1760.

    Article  PubMed  CAS  Google Scholar 

  22. Lee, D.N., and Landick, R. (1992). Structure of RNA and DNA chains in paused transcription complexes containing Escherichia coli RNA polymerase. J. Mol. Biol. 228, 759–777.

    Article  PubMed  CAS  Google Scholar 

  23. Carles-Kinch, K., and Kreuzer, K.N. (1997). RNA–DNA hybrid formation at bacteriophage T4 replication origin. J. Mol. Biol. 266, 915–926.

    PubMed  CAS  Google Scholar 

  24. Jeppesen, C., and Nielsen, P.E. (1988). Detection of intercalation-induced changes in DNA structure by reaction with diethyl pyrocarbonate or potassium permanganate. FEBS Lett. 231, 172–176.

    Article  PubMed  CAS  Google Scholar 

  25. Fox, K.R., and Grigg, G.W. (1988). Diethyl pyrocarbonate and permanganate provide evidence for an unusual DNA conformation induced by binding of the antitumour antibiotics bleomycin and phleomycin. Nucleic Acids Res. 16, 2063–2075.

    Article  PubMed  CAS  Google Scholar 

  26. Bailly, C., Gentle, D., Hamy, F., Purcell, M., and Waring, M.J. (1994). Localized chemical reactivity in DNA associated with the sequence-specific bisintercalation of echinomycin. Biochem. J. 300, 165–173.

    PubMed  CAS  Google Scholar 

  27. Michelottic, G.A., Michelotti, E.F., Pullner, A., Duncan, R.C., Eick, D., and Levens, D. (1996). Multiple single-stranded cis elements are associated with activated chromatin of the human c-myc gene in vivo. Mol. Cell. Biol. 16, 2656–2669.

    Google Scholar 

  28. Hershkovitz, M., and Riggs, A.D. (1997). Ligation-mediated PCR for chromatin-structure analysis of interphase and metaphase chromatin. Methods 11, 253–263.

    Article  PubMed  CAS  Google Scholar 

  29. Chiu, S.K., Rao, B.J., Story, R.M., and Radding, C.M. (1993). Interactions of three strands in joints made by RecA protein. Biochemistry 32, 13146–13155.

    Article  PubMed  CAS  Google Scholar 

  30. Voloshin, O.N., and Camerini-Otero, R.D. (1997). The duplex DNA is very underwound in the three-stranded RecA protein-mediated synaptic complex. Genes Cells 2, 303–314.

    Article  PubMed  CAS  Google Scholar 

  31. Plug, A.W., Peters, A.H.F.M., Keegan, K.S., Hoekstra, M.F., De Boer, P., and Ashley, T. (1998). Changes in protein composition of meiotic nodules during mammalian meiosis. J. Cell Sci. 111, 413–423.

    PubMed  CAS  Google Scholar 

  32. Duncan, R., Bazar, L., Michelotti, G., Tomonaga, T., Krutzch, H., Avigan, M., and Levens, D. (1994). A sequence-specific, single-strain binding protein activates the far upstream element of c-myc and defines a new DNA-binding motif. Genes Dev. 4, 465–480.

    Article  Google Scholar 

  33. Sun, W., and Godson, G.N. (1998). Structure of the Escherichia coli primase/single-strand DNA-binding protein/ phage G4ori-c complex required for primer RNA synthesis. J. Mol. Biol. 276, 689–703.

    Article  PubMed  CAS  Google Scholar 

  34. Godson, G.N., Mustaev, A.A., and Sun, W. (1998). ATP cross-linked to Escherichia coli single-strand DNA-binding protein can be utilized by the catalytic center of primase as initiating nucleotide for primer RNA synthesis on phage G4ori-c template. Biochemistry 37, 3810–3817.

    Article  PubMed  CAS  Google Scholar 

  35. McCarthy, J.G., and Rich, A. (1991). Detection of an unusual distortion in A-tract DNA using potassium permanganate effect of temperature and distamycin on the altered conformation. Nucleic Acids Res. 19, 3421–3430.

    Article  PubMed  CAS  Google Scholar 

  36. Matyasek, R., Fulnecek, J., Fajkus, J., and Bazdek, M. (1996). Evidence for a sequence-directed conformation periodicity in the genomic highly repetitive DNA detectable with single-strand-specific chemical probe potassium permanganate. Chromosome Res. 4, 340–349.

    Article  PubMed  CAS  Google Scholar 

  37. Epplen, J.T., Kyas, A., and Maeueler, W. (1996). Genomic simple repetitive DNAs are targets for differential binding of nuclear proteins. FEBS Lett. 389, 92–95.

    Article  PubMed  CAS  Google Scholar 

  38. Herr, W. (1985). Diethyl pyrocarbonate: A chemical probe for secondary structure in negatively supercoiled DNA. Proc. Natl Acad. Sci. USA 82, 8009–8013.

    Article  PubMed  CAS  Google Scholar 

  39. Voloshin, O.N., Mirkin, S.M., Lyamichev, V.I., Belotserkovskii, B.P., and Frank-Kamenetskii, M.D. (1988). Chemical probing of homopurine–homopyrimidine mirror repeats in supercoiled DNA. Nature 333, 475–476.

    Article  PubMed  CAS  Google Scholar 

  40. Bentin, T., and Nielsen, P.E. (1996). Enhanced peptide nucleic acid binding to supercoiled DNA: Possible implications for DNA “breathing” dynamics. Biochemistry 35, 8863–8869.

    Article  PubMed  CAS  Google Scholar 

  41. Furlong, J.C., and Lilley, D.M.J. (1986). Highly selective chemical modification of cruciform loops by diethyl pryrocarbonate. Nucleic Acids Res. 14, 3995–4007.

    Article  PubMed  CAS  Google Scholar 

  42. Scholten, P.M., and Nordheim, A. (1986). Diethyl pyrocarbonate: A chemical probe for DNA cruciforms. Nucleic Acids Res. 14, 3981–3993.

    Article  PubMed  CAS  Google Scholar 

  43. Balagurumoorthy, P., and Brahmachari, S.K. (1994). Structure and stability of human telomeric sequences. J. Biol. Chem. 269, 21858–21869.

    PubMed  CAS  Google Scholar 

  44. Nadel, Y., Weisman-Shomer, P., and Fry, M. (1995). The fragile X syndrome single strand d(CGG)n nucleotide repeats readily fold back to form unimolecular hairpin structures. J. Biol. Chem 270, 28970–28977.

    Article  PubMed  CAS  Google Scholar 

  45. Jiang, H., Zacharia, W., and Amirhaeri, S. (1991). Potassium permanganate as an in situ probe for B–Z and Z–Z junctions. Nucleic Acids Res. 19, 6943–6948.

    Article  PubMed  CAS  Google Scholar 

  46. Woelfl, S., Wittig, B., and Rich, A. (1995). Identification of transcriptionally induced Z-DNA segments in the human c-myc gene. Biochim. Biophys. Acta 1264, 294–302.

    Google Scholar 

  47. Glover, J.N.M., Farah, C.S., and Pulleyblank, D.E. (1990). Structural characterization of separated H-DNA conformers. Biochemistry 29, 11110–11115.

    Article  PubMed  CAS  Google Scholar 

  48. Haner, R., and Dervan, P.B. (1990). Single-strand DNA triple-helix formation. Biochemistry 29, 9761–9765.

    Article  PubMed  CAS  Google Scholar 

  49. Huibregtse, J.M., and Engelke, D.R. (1991). Direct sequence and footprint analysis of yeast DNA by primer extension. Methods Enzymol. 194, 550–562.

    Article  PubMed  CAS  Google Scholar 

  50. Holmes, D.S., and Quigley, M. (1981). A rapid boiling method for the preparation of bacterial plasmids. Anal. Biochem. 114, 193–197.

    Article  PubMed  CAS  Google Scholar 

  51. Owen, R.J., and Borman, P. (1987). A rapid biochemical method for purifying high molecular weight chromosomal DNA for restriction enzyme analysis. Nucleic Acids Res. 15, 3631.

    Article  PubMed  CAS  Google Scholar 

  52. Mueller, P.R., and Wold, B.J. (1989). In vivo footprinting of a muscle specific enhancer by ligation mediated PCR. Science 246, 780–786.

    Article  PubMed  CAS  Google Scholar 

  53. Garrity, P.A., and Wold, B.J. (1992). Effects of different DNA polymerases in ligation-mediated PCR: Enhanced genomic sequencing and in vivo footprinting. Proc. Natl Acad. Sci. USA 89, 1021–1025.

    Article  PubMed  CAS  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Department of Biochemistry and Molecular Biology, Colorado State University, 1870 Campus Delivery, Fort Collins, CO, 80523, USA

    Brenda F. Kahl & Marvin R. Paule

Authors
  1. Brenda F. Kahl
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Marvin R. Paule
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Marvin R. Paule .

Editor information

Editors and Affiliations

    Rights and permissions

    Reprints and permissions

    Copyright information

    © 2009 Humana Press, a part of Springer Science+Business Media, LLC

    About this protocol

    Cite this protocol

    Kahl, B.F., Paule, M.R. (2009). The Use of Diethyl Pyrocarbonate and Potassium Permanganate as Probes for Strand Separation and Structural Distortions in DNA. In: Leblanc, B., Moss, T. (eds) DNA-Protein Interactions. Methods in Molecular Biology™, vol 543. Humana Press. https://doi.org/10.1007/978-1-60327-015-1_6

    Download citation

    • DOI: https://doi.org/10.1007/978-1-60327-015-1_6

    • Published:

    • Publisher Name: Humana Press

    • Print ISBN: 978-1-60327-014-4

    • Online ISBN: 978-1-60327-015-1

    • eBook Packages: Springer Protocols

    Publish with us

    Policies and ethics

    Search

    Navigation