Molecular Biology

, Volume 52, Issue 4, pp 609–620 | Cite as

Ultrasonic Footprinting

  • S. L. Grokhovsky
Structural Functional Analysis Of Biopolymers and Their Complexes


Ligand binding influences the dynamics of the DNA helix in both the binding site and adjacent regions. This, in particular, is reflected in the changing pattern of cleavage of complexes under the action of ultrasound. The specificity of ultrasound-induced cleavage of the DNA sugar-phosphate backbone was studied in actinomycin D (AMD) complexes with double-stranded DNA restriction fragments. After antibiotic binding, the cleavage intensity of phosphodiester bonds between bases was shown to decrease at the chromophore intercalation site and to increase in adjacent positions. The character of cleavage depended on the sequences flanking the binding site and the presence of other AMD molecules bound in the close vicinity. A comparison of ultrasonic and DNase I cleavage patterns of AMD–DNA complexes provided more detail on the local conformation and dynamics of the DNA double helix in both binding site and adjacent regions. The results pave the way for developing a novel approach to studies of the nucleotide sequence dependence of DNA conformational dynamics and new techniques to identify functional genome regions.


actinomycin D footrpinting ultrasound DNA cleavage DNA conformation 



actinomycin D


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. 1.
    Waring M.J. 2006. Sequence-specific DNA Binding Agents. Ed. Waring M.J. Royal Soc. Chem. Biomol. Sci. Series.Google Scholar
  2. 2.
    Sarai A., Kono H. 2005. Protein–DNA recognition patterns and predictions. Ann. Rev. Biophys. Biomol. Struct. 34, 379–398.CrossRefGoogle Scholar
  3. 3.
    Burd J.F., Larson J.E., Wells R.D. 1975. Further studies on telestability in DNA. The synthesis and characterization of the duplex block polymers d(C20A10) · d(T10G20) and d(C20A15)-d(T15G20). J. Biol. Chem. 250, 6002–6007.PubMedGoogle Scholar
  4. 4.
    Burd J.F., Wartell R.M., Dodgson J.B., Wells R.D. 1975. Transmission of stability (telestability) in deoxyribonucleic acid. Physical and enzymatic studies on the duplex block polymer d(C15A15)•d(T15G15). J. Biol. Chem. 250, 5109–5113.PubMedGoogle Scholar
  5. 5.
    Olson W.K., Gorin A.A., Lu X.J., Hock L.M., Zhurkin V.B. 1998. DNA sequence-dependent deformability deduced from protein–DNA crystal complexes. Proc. Natl Acad. Sci. U. S. A. 95, 11163–11168.CrossRefPubMedPubMedCentralGoogle Scholar
  6. 6.
    Sims G.E., Kim S.H. 2003. Global mapping of nucleic acid conformational space: Dinucleoside monophosphate conformations and transition pathways among conformational classes. Nucleic Acids Res. 31, 5607–5616.CrossRefPubMedPubMedCentralGoogle Scholar
  7. 7.
    Svozil D., Kalina J., Omelka M., Schneider B. 2008. DNA conformations and their sequence preferences. Nucleic Acids Res. 36, 3690–3706.CrossRefPubMedPubMedCentralGoogle Scholar
  8. 8.
    Heddi B., Oguey C., Lavelle C., Foloppe N., Hartmann B. 2009. Intrinsic flexibility of B-DNA: The experimental TRX scale. Nucleic Acids Res. 38, 1034–1047.CrossRefPubMedPubMedCentralGoogle Scholar
  9. 9.
    Abi-Ghanem J., Heddi B., Foloppe N., Hartmann B. 2010. DNA structures from phosphate chemical shifts. Nucleic Acids Res. 38, e18.CrossRefPubMedGoogle Scholar
  10. 10.
    Fox K.R., Waring M.J. 1984. DNA structural variations produced by actinomycin and distamycin as revealed by DNAase I footpnatiag. Nucleic Acids Res. 12, 9271–9285.CrossRefPubMedPubMedCentralGoogle Scholar
  11. 11.
    Neidle S., Pearl L.H. Skelly J.V. 1987. DNA structure and perturbation by drug binding. Biochem. J. 243, 1–13.CrossRefPubMedPubMedCentralGoogle Scholar
  12. 12.
    Bullwinkle T.J., Koudelka G.B. 2011. The lysis-lysogeny decision of bacteriophage 933W: A 933W repressormediated long-distance loop has no role in regulating 933W PRM activity. J. Bacteriol. 193, 3313–3323.CrossRefPubMedPubMedCentralGoogle Scholar
  13. 13.
    Tullius T.D. 1988. DNA footprinting with hydroxyl radical. Nature. 332, 663–664.CrossRefPubMedGoogle Scholar
  14. 14.
    Van Dyke M.W., Dervan P.B. 1983. Footprinting with MPE·Fe(II). Complementary-strand analyses of distamycin- and actinomycin-binding sites on heterogeneous DNA. Cold Spring Harbor Symp. Quant Biol. 47, 347–353.CrossRefPubMedGoogle Scholar
  15. 15.
    Spassky A., Angelov D. 1997. Influence of the local helical conformation on the guanine modifications generated from one-electron DNA oxidation. Biochemistry. 36, 6571–6576.CrossRefPubMedGoogle Scholar
  16. 16.
    Vtyurina N.N., Grokhovsky S.L., Vasiliev A.B., Titov I.I., Ponomarenko P.M., Ponomarenko M.P., Peltek S.E., Nechipurenko Yu.D., Kolchanov N.A. 2012. Contextual DNA features significant for the DNA damage by the 193-nm ultraviolet laser beam. Dokl. Biochem. Biophys. 447, 267–272.CrossRefPubMedGoogle Scholar
  17. 17.
    Grokhovsky S.L., Zubarev V.E. 1991. Sequence-specific cleavage of double-stranded DNA caused by X-ray ionization of the platinum atom in the Pt-bis-netropsin–DNA complex. Nucleic Acids Res. 19, 257–264.CrossRefPubMedPubMedCentralGoogle Scholar
  18. 18.
    Galas D.J., Schmitz A. 1978. DNase footprinting: A simple method for the detection of protein–DNA binding specificity. Nucleic Acids Res. 5, 3157–3170.CrossRefPubMedPubMedCentralGoogle Scholar
  19. 19.
    Dervan P.B. 1986. Design of sequence-specific DNAbinding molecules. Science. 232, 464–471.CrossRefPubMedGoogle Scholar
  20. 20.
    Drew H.R. 1984. Structural specificities of five commonly used DNA nucleases. J. Mol. Biol. 176, 535–557.CrossRefPubMedGoogle Scholar
  21. 21.
    Suck D., Oefner C. 1986. Structure of DNase I at 2.0 Å resolution suggests a mechanism for binding to and cutting DNA. Nature. 321, 620–625.CrossRefPubMedGoogle Scholar
  22. 22.
    Skamrov A.V., Rybalkin I.N., Bibilashvili R.Sh., Gottikh B.P., Grokhovskii S.L., Gursky G.V., Zhuze A.L., Zasedatelev A.S., Nechipurenko D.Yu., Khorlin A.A. 1985. Specific protection of DNA by distamycin A, netropsin and bis-netropsins against the action of DNAse I. Mol. Biol. (Moscow). 19, 177–195.Google Scholar
  23. 23.
    Balasubramanian B., Pogozelski W.K., Tullius T.D. 1998. DNA strand breaking by the hydroxyl radical is governed by the accessible surface areas of the hydrogen atoms of the DNA backbone. Proc. Nat. Acad. Sci. U. S. A. 95, 9738–9743.CrossRefGoogle Scholar
  24. 24.
    Belikov S.V., Grokhovsky S.L., Isaguliants M.G., Surovaya A.N., Gursky G.V. 2005. Sequence-specific minor groove binding ligands as potential regulators of gene expression in Xenopus laevis oocytes. J. Biomol. Struct. Dynam. 23, 193–202.CrossRefGoogle Scholar
  25. 25.
    Wells R.D. 2009. Discovery of the role of non-B DNA structures in mutagenesis and human genomic disorders. J. Biol. Chem. 284, 8997–900.CrossRefPubMedPubMedCentralGoogle Scholar
  26. 26.
    Grokhovsky S. L. 2006. Specificity of DNA cleavage by ultrasound. Mol. Biol. (Moscow). 40, 276–283.CrossRefGoogle Scholar
  27. 27.
    Grokhovsky S.L., Il’icheva I.A., Nechipurenko D.Yu., Panchenko L.A., Polozov R.V., Nechipurenko Yu.D. 2008. Ultrasonic cleavage of DNA: Quantitative analysis of sequence specificity. Biophysics (Moscow). 53, 250–251.CrossRefGoogle Scholar
  28. 28.
    Grokhovsky S., Il’icheva I., Nechipurenko D., Golovkin M., Taranov G., Panchenko L., Polozov R., Nechipurenko Y. 2012. Quantitative analysis of electrophoresis data-application to sequence-specific ultrasonic cleavage of DNA. In: Gel Electrophoresis-Principles and Basics. London: InTech. doi 10.5772/2205Google Scholar
  29. 29.
    Das R., Laederach A., Pearlman S.M., Herschlag D., Altman R.B. 2005. SAFA: semi-automated footprinting analysis software for high-throughput quantification of nucleic acid footprinting experiments. RNA. 11, 344–354.CrossRefPubMedPubMedCentralGoogle Scholar
  30. 30.
    Nechipurenko Yu.D., Golovkin M.V., Nechipurenko D.Yu., Il’icheva I.N., Panchenko L.A., Polozov R.V., Grokhovsky S.L. 2008. Quantitative methods for analysis of DNA cleavage by ultrasound. Mathematics, Computer, Education: Proc. 15th Int. Conf. Izhevsk, vol. 3, pp. 26–35.Google Scholar
  31. 31.
    Grokhovsky S.L., Ilicheva I.A., Nechipurenko D.Yu., Golovkin M.V., Panchenko L.A., R.V. Polozov, Nechipurenko Y.D. 2011. Sequence-specific ultrasonic cleavage of DNA. Biophys. J. 100, 117–125.CrossRefPubMedPubMedCentralGoogle Scholar
  32. 32.
    Kang H.J., Park H.J. 2009. Novel molecular mechanism for actinomycin D activity as an oncogenic promoter G-quadruplex binder. Biochemistry. 48, 7392–7398.CrossRefPubMedGoogle Scholar
  33. 33.
    Brown D.R., Kurz M., Kearns D.R., Hsu V.L. 1994. Formation of multiple complexes between actinomycin D and a DNA hairpin: Structural characterization by multinuclear NMR. Biochemistry. 33, 651–664.CrossRefPubMedGoogle Scholar
  34. 34.
    Chen F.-M., Sha F., Chin K.-H., Chou S.-H. 2004. The nature of actinomycin D binding to d(AACCAXYG) sequence motifs. Nucleic Acids Res. 32, 271–277.CrossRefPubMedPubMedCentralGoogle Scholar
  35. 35.
    Rill R.L., Hecker K.H. 1996. Sequence-specific actinomycin D binding to single stranded DNA inhibits HIV reverse transcriptase and other polymerases. Biochemistry. 35, 3525–3533.CrossRefPubMedGoogle Scholar
  36. 36.
    Wadkins R.M., Vladu B. Tung C.S. 1998. AMD binds to metastable hairpins in single-stranded DNA. Biochemistry. 37, 11915–11923.CrossRefPubMedGoogle Scholar
  37. 37.
    Gallego J., Ortiz A.R., Pascual-Teresa B. Gago F. 1997. Structure–affinity relationships for the binding of actinomycin D to DNA. J. Comp.-Aid. Mol. Design. 11, 114–128.CrossRefGoogle Scholar
  38. 38.
    Nikitin S.M., Grokhovskii S.L., Zhuze A.L., Mikhailov M.V., Zasedatelev A.S., Gurskii G.V., Gottikh B.P 1981. Ligands possessing affinity for definite bases or base sequences of DNA: 5. Analogs of actinomycin D with substituents in position 7 of the phenoxazone chromophore. Russ. J. Bioorg. Chem. 7, 289–297.Google Scholar
  39. 39.
    Hampshirea A.J., Fox K.R. 2008. The effects of local DNA sequence on the interaction of ligands with their preferred binding sites. Biochimie. 90, 988–998.CrossRefGoogle Scholar
  40. 40.
    Bailly C., Marchand C., Waring M.J. 1993. New binding sites for antitumor antibiotics created by relocating the purine 2-amino group in DNA. J. Am. Chem. Soc. 115, 3784–3785.CrossRefGoogle Scholar
  41. 41.
    Bailly C., Graves D.E., Ridge G., Waring M.J. 1994. Use of a photoactive derivative of actinomycin to investigate shuffling between binding sites on DNA. Biochemistry. 33, 8736–8745.CrossRefPubMedGoogle Scholar
  42. 42.
    Qu X., Ren J., Riccelli P.V., Benight A.S., Chaires J.B. 2003. Enthalpy/entropy compensation: Influence of DNA flanking sequence on the binding of 7-amino actinomycin D to its primary binding site in short DNA duplexes. Biochemistry. 42, 11960–11967.CrossRefPubMedGoogle Scholar
  43. 43.
    Lian C.Y. Robinson H., Wang A.H.J. 1996. Structure of actinomycin D bound with (GAAGCTTC)2 and (GATGCTTC)2 and its binding to the (CAG)n: (CTG)n triplet sequence as determined by NMR analysis, J. Am. Chem. Soc. 118, 8791–8801.CrossRefGoogle Scholar
  44. 44.
    Kamitori S., Takusagawa F. 1992. Crystal structure of the 2:1 complex between d (GAAGCTTC) and the anticancer drug actinomycin D. J. Mol. Biol. 225, 445–456.CrossRefPubMedGoogle Scholar
  45. 45.
    Zhou N., James T. L., Shafer R. H. 1989. Binding of actinomycin D to [d (ATCGAT)]2: NMR evidence of multiple complexes. Biochemistry. 28, 5231–5239.CrossRefPubMedGoogle Scholar
  46. 46.
    Vekshin N.L. 2009. Biophysics of DNA–Actinomycin Nanocomplexes. Pushchino: Foton-Vek.Google Scholar
  47. 47.
    Sobell H.M., Jain S.C., Sakore T.D., Nordman C.E. 1971. Stereochemistry of actinomycin–DNA binding. Nature. 231, 200–205.Google Scholar
  48. 48.
    Crothers D.M., Müller W. 1973. Origins of base specificity in actinomycin and other DNA ligands. Cancer Chemother. Rep. 58, 97–100.Google Scholar
  49. 49.
    Rescifina A., Zagni C., Varrica M.G., Pistarà V., Corsaro A. 2014. Recent advances in small organic molecules as DNA intercalating agents: Synthesis, activity, and modeling. Eur. J. Med. Chem. 74, 95–115.CrossRefPubMedGoogle Scholar
  50. 50.
    Krugh T.R., Mooberry E.S., Chiao Y.C.C. 1977. Proton magnetic resonance studies of actinomycin D complexes with mixtures of nucleotides as models for the binding of the drug to DNA. Biochemistry. 16, 740–747.CrossRefPubMedGoogle Scholar
  51. 51.
    Paramanathan T., Vladescu I., McCauley M.J., Rouzina I., Williams M.C. 2012. Force spectroscopy reveals the DNA structural dynamics that govern the slow binding of actinomycin D. Nucleic Acids Res. 40, 4925–4932.CrossRefPubMedPubMedCentralGoogle Scholar
  52. 52.
    Scamrov A.V., Beabealashvilli R.S. 1983. Binding of actinomycin D to DNA revealed by DNase I footprinting. FEBS Lett. 164, 97–101.CrossRefPubMedGoogle Scholar
  53. 53.
    Waterloh K., Fox K.R. 1992. Secondary (non-GpC) binding sites for actinomycin on DNA. Biochim. Biophys. Acta. 1131, 300–306.CrossRefPubMedGoogle Scholar
  54. 54.
    Chen F.-M. 1992. Binding specificities of actinomycin D to non-self-complementary-XGCY-tetranucleotide sequences. Biochemistry. 31, 6223–6228.CrossRefPubMedGoogle Scholar
  55. 55.
    Bailey S.A., Graves D.E., Rill R., Marsch G. 1993. Influence of DNA base sequence on the binding energetics of actinomycin D. Biochemistry. 32, 5881–5887.CrossRefPubMedGoogle Scholar
  56. 56.
    Fletcher M.C., Fox K.R. 1996. Dissociation kinetics of actinomycin D from individual GpC sites in DNA. Eur. J. Biochem. 237, 164–170.CrossRefPubMedGoogle Scholar
  57. 57.
    Fox K.R., Waring M.J. 1984. DNA structural variations produced by actinomycin and distamycin as revealed by DNase I footprinting. Nucleic Acids Res. 12, 9271–9285.CrossRefPubMedPubMedCentralGoogle Scholar
  58. 58.
    Hogan M.E., Roberson M.W., Austin R.H. 1989. DNA flexibility variation may dominate DNase I cleavage. Proc. Nat. Acad. Sci. U. S. A. 86, 9273–9277.CrossRefGoogle Scholar
  59. 59.
    Brukner I., Sanchez R., Suck D., Pongor S. 1995. Sequence-dependent bending propensity of DNA as revealed by DNase I: Parameters for trinucleotides. EMBO J. 14, 1812.PubMedPubMedCentralCrossRefGoogle Scholar
  60. 60.
    Basedow A.M., Ebert E.B. 1977. Ultrasonic degradation of polymers in solution. In: Advances in Polymers Science. Eds. Abe A., Albertsson A.-C., Genzer J. Berlin: Springer, vol. 22, pp. 83–148.CrossRefGoogle Scholar
  61. 61.
    Scott E.V., Zon G., Marzilli L.G., Wilson W.D. 1988. 2D NMR investigation of the binding of the anticancer drug actinomycin D to duplexed dATGCGCAT: Conformational features of the unique 2:1 adduct. Biochemistry. 27, 7940–7951.CrossRefPubMedGoogle Scholar
  62. 62.
    Chen H., Liu X., Patel D.J. 1996. DNA bending and unwinding associated with actinomycin D antibiotics bound to partially overlapping sites on DNA. J. Mol. Biol. 258, 457–479.CrossRefPubMedGoogle Scholar
  63. 63.
    Il’icheva I.A., Nechipurenko D.Yu., Grokhovsky S.L. 2009. Ultrasonic cleavage of nicked DNA. J. Biomol. Struct. Dynam. 27, 391–398.CrossRefGoogle Scholar
  64. 64.
    Huang M., Giese T.J., Lee T.S., York D.M. 2014. Improvement of DNA and RNA sugar pucker profiles from semiempirical quantum methods. J. Chem. Theory Comput. 10, 1538–1545.CrossRefPubMedPubMedCentralGoogle Scholar
  65. 65.
    Todd R.C., Lippard S.J. 2010. Structure of duplex DNA containing the cisplatin 1, 2-Pt (NH 3. 2 2+- d(GpG) cross-link at 1.77 Å resolution. J. Inorg. Biochem. 104, 902–908.CrossRefPubMedPubMedCentralGoogle Scholar
  66. 66.
    Liu X., Chen H., Patel D.J. 1991. Solution structure of actinomycin-DNA complexes: Drug intercalation at isolated GCsites. J. Biomol. NMR. 1, 323–347.CrossRefPubMedGoogle Scholar

Copyright information

© Pleiades Publishing, Inc. 2018

Authors and Affiliations

  1. 1.Engelhardt Institute of Molecular BiologyRussian Academy of SciencesMoscowRussia

Personalised recommendations