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Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 1985 Dec;82(23):8009–8013. doi: 10.1073/pnas.82.23.8009

Diethyl pyrocarbonate: a chemical probe for secondary structure in negatively supercoiled DNA.

W Herr
PMCID: PMC391431  PMID: 3865212

Abstract

Purine residues located within regions of DNA that have the potential to form left-handed Z-helical structures are modified preferentially by diethyl pyrocarbonate; this hyperreactivity is dependent on the degree of negative superhelicity of the circular DNA molecules. As negative superhelical density increases, guanosines in a 32-base-pair alternating G-C sequence and adenosines (but not guanosines) in a 64-base-pair alternating A-C/G-T sequence become 5- to 10-fold more reactive to diethyl pyrocarbonate. The negative superhelical densities at which enhanced reactivity occurs are similar to those reported for the point at which left-handed helices form within plasmids carrying these DNA sequences. Probing of negatively supercoiled pBR322 with diethyl pyrocarbonate reveals a hyperreactive region 31 base pairs in length of which only 9 base pairs are a perfect alternating purine and pyrimidine sequence; the reactivity of purines within this sequence indicates that purines in the anti conformation, or guanosines in the syn conformation with neighboring 3' thymidines, are not hyperreactive in the Z-DNA form.

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Selected References

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  1. Azorin F., Nordheim A., Rich A. Formation of Z-DNA in negatively supercoiled plasmids is sensitive to small changes in salt concentration within the physiological range. EMBO J. 1983;2(5):649–655. doi: 10.1002/j.1460-2075.1983.tb01479.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Di Capua E., Stasiak A., Koller T., Brahms S., Thomae R., Pohl F. M. Torsional stress induces left-handed helical stretches in DNA of natural base sequence: circular dichroism and antibody binding. EMBO J. 1983;2(9):1531–1535. doi: 10.1002/j.1460-2075.1983.tb01619.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Haniford D. B., Pulleyblank D. E. Facile transition of poly[d(TG) x d(CA)] into a left-handed helix in physiological conditions. Nature. 1983 Apr 14;302(5909):632–634. doi: 10.1038/302632a0. [DOI] [PubMed] [Google Scholar]
  4. Haschemeyer A. E., Rich A. Nucleoside conformations: an analysis of steric barriers to rotation about the glycosidic bond. J Mol Biol. 1967 Jul 28;27(2):369–384. doi: 10.1016/0022-2836(67)90026-5. [DOI] [PubMed] [Google Scholar]
  5. Herr W., Corbin V., Gilbert W. Nucleotide sequence of the 3' half of AKV. Nucleic Acids Res. 1982 Nov 11;10(21):6931–6944. doi: 10.1093/nar/10.21.6931. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Ish-Horowicz D., Burke J. F. Rapid and efficient cosmid cloning. Nucleic Acids Res. 1981 Jul 10;9(13):2989–2998. doi: 10.1093/nar/9.13.2989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Johnston B. H., Rich A. Chemical probes of DNA conformation: detection of Z-DNA at nucleotide resolution. Cell. 1985 Oct;42(3):713–724. doi: 10.1016/0092-8674(85)90268-5. [DOI] [PubMed] [Google Scholar]
  8. Keller W. Determination of the number of superhelical turns in simian virus 40 DNA by gel electrophoresis. Proc Natl Acad Sci U S A. 1975 Dec;72(12):4876–4880. doi: 10.1073/pnas.72.12.4876. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Leonard N. J., McDonald J. J., Henderson R. E., Reichmann M. E. Reaction of diethyl pyrocarbonate with nucleic acid components. Adenosine. Biochemistry. 1971 Aug 31;10(18):3335–3342. doi: 10.1021/bi00794a003. [DOI] [PubMed] [Google Scholar]
  10. Maxam A. M., Gilbert W. A new method for sequencing DNA. Proc Natl Acad Sci U S A. 1977 Feb;74(2):560–564. doi: 10.1073/pnas.74.2.560. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Maxam A. M., Gilbert W. Sequencing end-labeled DNA with base-specific chemical cleavages. Methods Enzymol. 1980;65(1):499–560. doi: 10.1016/s0076-6879(80)65059-9. [DOI] [PubMed] [Google Scholar]
  12. Nordheim A., Lafer E. M., Peck L. J., Wang J. C., Stollar B. D., Rich A. Negatively supercoiled plasmids contain left-handed Z-DNA segments as detected by specific antibody binding. Cell. 1982 Dec;31(2 Pt 1):309–318. doi: 10.1016/0092-8674(82)90124-6. [DOI] [PubMed] [Google Scholar]
  13. Nordheim A., Rich A. Negatively supercoiled simian virus 40 DNA contains Z-DNA segments within transcriptional enhancer sequences. Nature. 1983 Jun 23;303(5919):674–679. doi: 10.1038/303674a0. [DOI] [PubMed] [Google Scholar]
  14. Nordheim A., Rich A. The sequence (dC-dA)n X (dG-dT)n forms left-handed Z-DNA in negatively supercoiled plasmids. Proc Natl Acad Sci U S A. 1983 Apr;80(7):1821–1825. doi: 10.1073/pnas.80.7.1821. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Peattie D. A., Gilbert W. Chemical probes for higher-order structure in RNA. Proc Natl Acad Sci U S A. 1980 Aug;77(8):4679–4682. doi: 10.1073/pnas.77.8.4679. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Peck L. J., Nordheim A., Rich A., Wang J. C. Flipping of cloned d(pCpG)n.d(pCpG)n DNA sequences from right- to left-handed helical structure by salt, Co(III), or negative supercoiling. Proc Natl Acad Sci U S A. 1982 Aug;79(15):4560–4564. doi: 10.1073/pnas.79.15.4560. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Peck L. J., Wang J. C. Energetics of B-to-Z transition in DNA. Proc Natl Acad Sci U S A. 1983 Oct;80(20):6206–6210. doi: 10.1073/pnas.80.20.6206. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Rich A., Nordheim A., Wang A. H. The chemistry and biology of left-handed Z-DNA. Annu Rev Biochem. 1984;53:791–846. doi: 10.1146/annurev.bi.53.070184.004043. [DOI] [PubMed] [Google Scholar]
  19. Singleton C. K., Klysik J., Stirdivant S. M., Wells R. D. Left-handed Z-DNA is induced by supercoiling in physiological ionic conditions. Nature. 1982 Sep 23;299(5881):312–316. doi: 10.1038/299312a0. [DOI] [PubMed] [Google Scholar]
  20. Vincze A., Henderson R. E., McDonald J. J., Leonard N. J. Reaction of diethyl pyrocarbonate with nucleic acid components. Bases and nucleosides derived from guanine, cytosine, and uracil. J Am Chem Soc. 1973 Apr 18;95(8):2677–2682. doi: 10.1021/ja00789a045. [DOI] [PubMed] [Google Scholar]
  21. Wang A. H., Gessner R. V., van der Marel G. A., van Boom J. H., Rich A. Crystal structure of Z-DNA without an alternating purine-pyrimidine sequence. Proc Natl Acad Sci U S A. 1985 Jun;82(11):3611–3615. doi: 10.1073/pnas.82.11.3611. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Wang A. H., Hakoshima T., van der Marel G., van Boom J. H., Rich A. AT base pairs are less stable than GC base pairs in Z-DNA: the crystal structure of d(m5CGTAm5CG). Cell. 1984 May;37(1):321–331. doi: 10.1016/0092-8674(84)90328-3. [DOI] [PubMed] [Google Scholar]
  23. Wang A. H., Quigley G. J., Kolpak F. J., Crawford J. L., van Boom J. H., van der Marel G., Rich A. Molecular structure of a left-handed double helical DNA fragment at atomic resolution. Nature. 1979 Dec 13;282(5740):680–686. doi: 10.1038/282680a0. [DOI] [PubMed] [Google Scholar]

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