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.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
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.
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.
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.
Hayatsu, H., and Ukita, T. (1967). The selective degredation of pyrimidines in nucleic acids by permanganate oxidation. Biochem. Biophys. Res. Commun 29, 556–561.
Howgate, P., Jones, A.S., and Tittensor, J.J. (1968). The permanganate oxidation of thymidine. J. Chem. Soc. C 1, 275–279.
Iida, S., and Hayatsu, H. (1971). The permanganate oxidation of thymidine. J. Biophys. Acta 240, 370–375.
Rubin, C.M., and Schmid, C.W. (1980). Pyrimidine-specific chemical reactions useful for DNA sequencing. Nucleic Acids Res. 8, 4613–4619.
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.
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.
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.
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.
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.
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.
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.
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.
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.
Kainz, M., and Roberts, J. (1992). Structures of transcription elongation complexes in vivo. Science 255, 838–841.
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.
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.
Hartvig, L., and Christiansen, J. (1996). Intrinsic termination of T7 RNA polymerase mediated by either RNA or DNA. EMBO J. 15, 4767–4774.
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.
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.
Carles-Kinch, K., and Kreuzer, K.N. (1997). RNA–DNA hybrid formation at bacteriophage T4 replication origin. J. Mol. Biol. 266, 915–926.
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.
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.
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.
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.
Hershkovitz, M., and Riggs, A.D. (1997). Ligation-mediated PCR for chromatin-structure analysis of interphase and metaphase chromatin. Methods 11, 253–263.
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.
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.
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.
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.
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.
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.
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.
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.
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.
Herr, W. (1985). Diethyl pyrocarbonate: A chemical probe for secondary structure in negatively supercoiled DNA. Proc. Natl Acad. Sci. USA 82, 8009–8013.
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.
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.
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.
Scholten, P.M., and Nordheim, A. (1986). Diethyl pyrocarbonate: A chemical probe for DNA cruciforms. Nucleic Acids Res. 14, 3981–3993.
Balagurumoorthy, P., and Brahmachari, S.K. (1994). Structure and stability of human telomeric sequences. J. Biol. Chem. 269, 21858–21869.
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.
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.
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.
Glover, J.N.M., Farah, C.S., and Pulleyblank, D.E. (1990). Structural characterization of separated H-DNA conformers. Biochemistry 29, 11110–11115.
Haner, R., and Dervan, P.B. (1990). Single-strand DNA triple-helix formation. Biochemistry 29, 9761–9765.
Huibregtse, J.M., and Engelke, D.R. (1991). Direct sequence and footprint analysis of yeast DNA by primer extension. Methods Enzymol. 194, 550–562.
Holmes, D.S., and Quigley, M. (1981). A rapid boiling method for the preparation of bacterial plasmids. Anal. Biochem. 114, 193–197.
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.
Mueller, P.R., and Wold, B.J. (1989). In vivo footprinting of a muscle specific enhancer by ligation mediated PCR. Science 246, 780–786.
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.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights 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