Biochemistry (Moscow)

, Volume 83, Issue 10, pp 1231–1244 | Cite as

Thermodynamic Study of Interactions of Distamycin A with Chromatin in Rat Liver Nuclei in the Presence of Polyamines

  • A. N. PrusovEmail author
  • T. A. Smirnova
  • G. Ya. Kolomijtseva


We studied the thermodynamics of melting of isolated rat liver nuclei with different degrees of chromatin condensation determined by the concentration of polyamines (PA) and the solution ionic strength, as well as the effect of the antibiotic distamycin A (DM) on melting. Differential scanning calorimetry (DSC) profiles of nuclear preparations contained three peaks that reflected melting of three main chromatin domains. The number of peaks did not depend on the degree of condensation; however, nuclei with more condensed chromatin had a higher total enthalpy. DM stabilized peaks II and III corresponding to the melting of relaxed and topologically strained DNA, respectively, but destabilized peak I corresponding to the melting of nucleosome core histones. At the saturating concentration (DM/DNA molar ratio = 0.1), DM increased Tm of peaks II and III by ~5°C and decreased Tm of peak I by ~2.5°C. Based on the dependence of ΔH on DM concentration, we established that at low DM/DNA ratio (⩽0.03), when DM interacted predominantly with AT-rich DNA regions, the enthalpy of peak II decreased in parallel with the increase in the enthalpy of peak III, which indicated that DM induces structural transitions in the nuclear chromatin associated with the increase in torsional stress in DNA. An increase in free energy under saturation conditions was equal to the change in the free energy of DM interaction with DNA. However, the increase in the enthalpy of melting of the nuclei in the presence of DM was much greater than the enthalpy of titration of nuclei with DM. This indicates a significant increase in the strength of interaction between the two DNA strands apparently due, among other things, to changes in the torsional stress of DNA in the nuclei. Titration of the nuclei with increasing PA concentrations resulted in the decrease in the number of DM-binding sites and the non-monotonous dependence of the enthalpy and entropy contribution to the binding free energy on the PA content. We suggested that the observed differences in the thermodynamic parameters were due to the different width of the minor groove in the nuclear chromatin DNA, which depends on PA concentration.


nucleus chromatin distamycin A differential scanning calorimetry isothermal calorimetric titration polyamines DNA torsional stress 



distamycin A


differential scanning calorimetry


isothermal titration calorimetry








melting temperature


thermodynamic parameters


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  1. 1.
    Jelesarov, I., and Bosshard, H. R. (1999) Isothermal titration calorimetry and differential scanning as complementary tools to investigate the energetics of biomolecular recognition, J. Mol. Recognit., 12, 3–18.CrossRefPubMedGoogle Scholar
  2. 2.
    Weber, P. C., and Salemme, F. R. (2003) Applications of calorimetric methods to drug discovery and the study of protein interactions, Curr. Opin. Struct. Biol., 13, 115–121.CrossRefPubMedGoogle Scholar
  3. 3.
    Almagor, M., and Cole, R. D. (1989) Differential scanning calorimetry of nuclei as a test for the effects of anticancer drugs on human chromatin, Cancer Res., 49, 5561–5566.PubMedGoogle Scholar
  4. 4.
    Aaronson, R. P., and Woo, E. (1981) Organization in the cell nucleus: divalent cations modulate the distribution of condensed and diffuse chromatin, J. Cell Biol., 90, 181–186.CrossRefPubMedGoogle Scholar
  5. 5.
    Schnell, S., and Hancock, R. (2008) The intranuclear environment, Methods Mol. Biol., 463, 3–19.CrossRefPubMedGoogle Scholar
  6. 6.
    Touchette, N. A., and Cole, R. D. (1985) Differential scan-ning calorimetry of nuclei reveals the loss of major structural features in chromatin by brief nuclease treatment, Proc. Natl. Acad. Sci. USA, 82, 2642–2646.CrossRefPubMedGoogle Scholar
  7. 7.
    Balbi, C., Abelmoschi, M. L., Gogioso, L., Parodi, S., Cavazza, B., and Patrone, E. (1989) Structural domains and conformational changes in nuclear chromatin: a quantitative thermodynamic approach by differential scanning calorimetry, Biochemistry, 28, 3220–3227.CrossRefPubMedGoogle Scholar
  8. 8.
    Cavazza, B., Brizzolara, G., Lazzarini, G., Patrone, E., Piccardo, M., Barboro, P., Parodi, S., Pasini, A., and Balbi, C. (1991) Thermodynamics of condensation of nuclear chromatin a differential scanning calorimetry study of the salt-dependent structural transitions, Biochemistry, 30, 9060–9072.CrossRefPubMedGoogle Scholar
  9. 9.
    Labarbe, R., Flock, S., Maus, C., and Houssier, C. (1996) Polyelectrolyte counterion condensation theory explains differential scanning calorimetry studies of salt-induced condensation of chicken erythrocyte chromatin, Biochemistry, 12, 3319–3327.CrossRefGoogle Scholar
  10. 10.
    Almagor, M., and Cole, R. D. (1989) Changes in chromatin structure during the aging of cell cultures as revealed by differential scanning calorimetry, Biochemistry, 28, 5686–5688.CrossRefGoogle Scholar
  11. 11.
    Almagor, M., and Cole, R. D. (1989) In physiological salt conditions the core proteins of the nucleosomes in large chromatin fragments denature at 73°C and the DNA unstacks at 85°C, J. Biol. Chem., 264, 6515–6519.PubMedGoogle Scholar
  12. 12.
    Touchette, N. A., and Cole, R. D. (1992) Effects of salt concentration and H1 histone removal on the DSC of nuclei, Biochemistry, 31, 1842–1849.CrossRefPubMedGoogle Scholar
  13. 13.
    Miller-Fleming, L., Olin-Sandoval, V., Campbell, K., and Ralser, M. (2015) Remaining mysteries of molecular biology: the role of polyamines in the cell, J. Mol. Biol., 427, 3389–3406.CrossRefPubMedGoogle Scholar
  14. 14.
    Visvanathan, A., Ahmed, K., Even-Faitelson, L., Lleres, D., Bazett-Jones, D. P., and Lamond, A. I. (2013) Modulation of higher order chromatin conformation in mammalian cell nuclei can be mediated by polyamines and divalent cations, PLoS One, 8, e67689.CrossRefPubMedPubMedCentralGoogle Scholar
  15. 15.
    Burgoyne, L. A., Wagar, M. A., and Atkinson, M. R. (1970) Calcium-dependent priming of DNA synthesis in isolated rat liver nuclei, Biochem. Biophys. Res. Commun., 39, 254–259.CrossRefPubMedGoogle Scholar
  16. 16.
    Nowotarski, S. L., Woster, P. M., and Casero, R. A., Jr. (2013) Polyamines and cancer: implications for chemotherapy and chemoprevention, Expert Rev. Mol. Med., 15, e3.CrossRefPubMedPubMedCentralGoogle Scholar
  17. 17.
    Nelson, S. M., Ferguson, L. R., and Denny, W. A. (2007) Non-covalent ligand–DNA interactions: minor groove binding agents, Mutat. Res., 623, 24–40.CrossRefPubMedGoogle Scholar
  18. 18.
    Lah, J., and Vesnaver, G. (2000) Binding of distamycin A and netropsin to the 12mer DNA duplexes containing mixed AT·GC sequences with at most five or three successive AT base pairs, Biochemistry, 39, 9317–9326.CrossRefPubMedGoogle Scholar
  19. 19.
    Majumber, P., and Dasgupta, D. (2011) Effect of DNA groove binder distamycin A upon chromatin structure, PLoS One, 6, e26486.CrossRefGoogle Scholar
  20. 20.
    Marverti, G., Guaitoli, G., Ligabue, A., Frassineti, Ch., Monti, M. G., Lombardi, P., and Costi, M. P. (2012) Distamycin A and derivatives as synergic drugs in cisplatin-sensitive and -resistant ovarian cancer cells, Amino Acids, 42, 641–653.CrossRefPubMedGoogle Scholar
  21. 21.
    Prusov, A. N., Smirnova, T. A., Kurochkina, L. P., and Kolomijtseva, G. Y. (2010) Influence of distamycin, chro-momycin, and UV-irradiation on extraction of histone H1 from rat liver nuclei by polyglutamic acid, Biochemistry (Moscow), 75, 1331–1341.Google Scholar
  22. 22.
    Gasser, S. M., Laroche, T., Falquet, J., Boy de la Tour, E., and Laemmli, U. K. (1986) Metaphase chromosome structure. Involvement of topoisomerase II, J. Mol. Biol., 188, 613–629.CrossRefPubMedGoogle Scholar
  23. 23.
    Kas, E., Poljak, L., Adachi, Y., and Laemmli, U. K. (1993) A model for chromatin opening: stimulation of topoiso-merase II and restriction enzyme cleavage of chromatin by distamycin, EMBO J., 12, 115–126.CrossRefPubMedPubMedCentralGoogle Scholar
  24. 24.
    Spirin, A. S. (1958) Spectrophotometric determination of total nucleic acids, Biokhimiia, 23, 656–662.PubMedGoogle Scholar
  25. 25.
    Schaffner, W., and Weismann, C. (1973) A rapid sensitive and specific method for the determination of protein in dilute solution, Analytic. Biochem., 56, 502–514.CrossRefGoogle Scholar
  26. 26.
    Dasgupta, D., Parrack, S. K., and Sasikekharan, V. (1987) Interaction of synthetic analogues of distamycin with poly(dA-dT): role of the conjugated N-methylpyrrole system, Biochemistry, 26, 6381–6386.CrossRefPubMedGoogle Scholar
  27. 27.
    Lepock, J. R., Frey, H. E., Heynen, M. L., Senisterra, G. A., and Warters, R. L. (2001) The nuclear matrix is a ther-molabile cellular structure, Cell Stress Chaperones, 6, 136–147.CrossRefPubMedPubMedCentralGoogle Scholar
  28. 28.
    Tiktopulo, E. I., Privalov, P. L., Odintsova, T. I., Ermokhina, T. M., Krasheninnikov, I. A., Aviles, F. X., Cary, P. D., and Crane-Robinson, C. (1982) The central tryptic fragment of histones H1 and H5 is a fully compacted domain and is the only folded region in the polypeptide chain. A thermody-namic study, Eur. J. Biochem., 122, 327–331.CrossRefPubMedGoogle Scholar
  29. 29.
    Lyubarev, A. E., and Kurganov, B. I. (2000) Investigation of irreversible denaturation of proteins using differential scamming calorimetry, Usp. Biol. Khim., 40, 43–84.Google Scholar
  30. 30.
    Kopka, M. L., Yoon, Ch., Goodsell, D., Pjura, Ph., and Dickerson, R. E. (1985) The molecular origin of DNA-drug specificity in netropsin and distamycin, Proc. Natl. Acad. Sci. USA, 82, 1376–1380.CrossRefPubMedGoogle Scholar
  31. 31.
    Van Dyke, M. W., Hertzberg, R. P., and Dervan, P. B. (1982) Map of distamycin, netropsin and actinomycin binding sites on heterogeneous DNA. DNA cleavage inhibition patterns with methidiumpropyl-EDTA·Fe(II), Proc. Natl. Acad. Sci. USA, 79, 5470–5474.CrossRefPubMedGoogle Scholar
  32. 32.
    Rao, K. E., Dasgupta, D., and Sasisekharan, V. (1988) Interaction of synthetic analogues of distamycin and netropsin with nucleic acids. Does curvature of ligand play a role in distamycin–DNA interactions, Biochemistry, 27, 3018–3024.CrossRefPubMedGoogle Scholar
  33. 33.
    Chaires, J. B. (1997) Energetics of drug–DNA interac-tions, Biopolymers, 44, 201–215.CrossRefPubMedGoogle Scholar
  34. 34.
    Record, M. T., Anderson, C. F., and Lohman, T. M. (1978) Thermodynamic analysis of ion effects on the binding and conformational equilibria of proteins and nucleic acids: the roles of ion association or release, screening, and ion effects on water activity, Quart. Rev. Biophys., 11, 103–178.CrossRefGoogle Scholar
  35. 35.
    Krylov, A. S., Grokhovsky, S. L., Zasedatelev, A. S., Zhuze, A. L., Gursky, G. V., and Gottikh, B. P. (1979) Quantitative estimation of the contribution of pyrrolcarboxamide groups of the antibiotic distamycin A into specificity of its binding to DNA ATpairs, Nucleic Acids Res., 6, 289–304.CrossRefPubMedPubMedCentralGoogle Scholar
  36. 36.
    Fagan, P., and Wemmer, D. E. (1992) Cooperative binding of distamycin A to DNA in the 2–1 mode, J. Am. Chem. Soc., 114, 1080–1081.CrossRefGoogle Scholar
  37. 37.
    Wemmer, D. E. (2000) Designed sequence-specific minor groove ligands, Annu. Rev. Biophys. Biomol. Struct., 29, 439–461.CrossRefPubMedGoogle Scholar
  38. 38.
    Olsen, E. A., Louie, E. A., Drobny, G. P., and Sigurdsson, S. T. (2003) Determination of DNA minor groove width in distamycin–DNA complexes by solid-state NMR, Nucleic Acids Res., 31, 5084–5089.CrossRefPubMedPubMedCentralGoogle Scholar
  39. 39.
    Dattagupta, N., Hogan, M., and Crothers, D. M. (1980) Interaction of netropsin and distamycin with deoxyribonucleic acid: electric dichroism study, Biochemistry, 19, 5998–6005.CrossRefPubMedGoogle Scholar
  40. 40.
    Fitzgerald, D. J., and Anderson, J. N. (1999) Selective nucleosome disruption by drugs that bind in the minor groove of DNA, J. Biol. Chem., 274, 27128–27138.CrossRefPubMedGoogle Scholar
  41. 41.
    Brown, P. M., and Fox, K. R. (1996) Minor groove binding ligands alter the rotational positioning of DNA on nucleosome core particles, J. Mol. Biol., 262, 671–685.CrossRefPubMedGoogle Scholar
  42. 42.
    Storl, K., Burckhardt, G., Lown, J. W., and Zimmer, C. (1993) Studies on the ability of minor groove binders to induce supercoiling in DNA, FEBS Lett., 334, 49–54.CrossRefPubMedGoogle Scholar
  43. 43.
    Prusov, A. N., Kolomijtseva, G. Ya., and Smirnova, T. A. (2017) Differential scanning calorimetric study of antibiotic distamycin A binding with chromatin within isolated rat liver nuclei, Pharm. Biol., 55, 687–690.CrossRefPubMedGoogle Scholar
  44. 44.
    Breslauer, K. J., Remeta, D. P., Chou, W.-Y., Ferrante, R., Curry, J., Zannczkowski, D., Snyder, J. G., and Marky, L. A. (1987) Enthalpy-entropy compensations in drug-DNA binding studies, Proc. Natl. Acad. Sci. USA, 84, 8922–8926.CrossRefPubMedGoogle Scholar
  45. 45.
    Majumber, P., Banerjee, A., Shandilya, J., Senapati, P., Chattejee, S., Kundu, T. K., and Dasgupta, D. (2013) Minor groove binder distamycin remodels chromatin but inhibits transcription, PLoS One, 8, e57693.CrossRefGoogle Scholar
  46. 46.
    Cerofolini, L., Amatob, J., Borsia, V., Paganob, B., Randazzob, A., and Fragaic, M. (2015) Probing the inter-action of distamycin A with S100β: the unexpected ability of S100β to bind to DNA-binding ligands, J. Mol. Recognit., 28, 376–384.CrossRefPubMedGoogle Scholar
  47. 47.
    Kas, E., Izaurralde, E., and Laemmli, U. K. (1989) Specific inhibition of DNA binding to nuclear scaffolds and histone H1 by distamycin. The role of oligo(dA)·oligo(dT) tracts, J. Mol. Biol., 210, 587–599.CrossRefPubMedGoogle Scholar
  48. 48.
    Marx, K. A., Zhou, Y., and Kishawi, I. (2006) Evidence for long poly(dA)·poly(dT) tracts in D. discoideum DNA at high frequencies and their preferential avoidance of nucle-osomal DNA core regions, J. Biomol. Struct. Dyn., 23, 429–446.CrossRefPubMedGoogle Scholar
  49. 49.
    Waldron, T. T., and Murphy, K. P. (2003) Stabilization of proteins by ligand binding: application to drug screening and determination of unfolding energetics, Biochemistry, 42, 5058–5064.CrossRefPubMedGoogle Scholar
  50. 50.
    Marky, L. A., Blumenfeld, K. S., and Breslauer, K. J. (1983) Calorimetric and spectroscopic investigation of drug–DNA interaction: I. The binding of netropsin to poly(dAT), Nucleic Acids Res., 11, 2857–2870.CrossRefPubMedPubMedCentralGoogle Scholar
  51. 51.
    Korolev, N., Lyubartsev, A. P., and Nordenskiold, L. (2010) Cation-induced polyelectrolyte–polyelectrolyte attraction in solutions of DNA and nucleosome core particles, Adv. Colloid Interface Sci., 158, 32–47.CrossRefPubMedGoogle Scholar
  52. 52.
    Marquet, R., Colson, P., and Houssier, C. (1986) The condensation of chromatin and histone H1-depleted chromatin by spermine, J. Biomol. Struct. Dyn., 4, 205–218.CrossRefPubMedGoogle Scholar
  53. 53.
    Deng, H., Bloomfield, V. A., Benevides, J. M., and Thomas, G. J., Jr. (2000) Structural basis of polyamine–DNA recognition: spermidine and spermine interactions with genomic B-DNAs of different GCcontent probed by Raman spec-troscopy, Nucleic Acids Res., 28, 3379–3385.CrossRefPubMedPubMedCentralGoogle Scholar
  54. 54.
    Schmidt, N., and Behr, J.-P. (1991) Location of spermine and other polyamines on DNA as revealed by photoaffinity cleavage with polyaminobenzenediazonium salts, Biochemistry, 30, 4357–4361.CrossRefGoogle Scholar
  55. 55.
    Kabir, A., and Kumar, G. S. (2013) Binding of the biogenic polyamines to deoxyribonucleic acids of varying base com-position: base specificity and the associated energetics of the interaction, PLoS One, 8, e70510.CrossRefPubMedPubMedCentralGoogle Scholar
  56. 56.
    Marquet, R., and Houssier, C. (1988) Different binding modes of spermine to A–T and G–C base pair modulate the bending and stiffening of the DNA double helix, J. Biomol. Struct. Dyn., 6, 235–246.CrossRefPubMedGoogle Scholar
  57. 57.
    Jen-Jacobson, L., Engler, L. E., and Jacobson, L. A. (2000) Structural and thermodynamic strategies for site-specific DNA binding proteins, Structure, 8, 1015–1023.CrossRefPubMedGoogle Scholar

Copyright information

© Pleiades Publishing, Ltd. 2018

Authors and Affiliations

  • A. N. Prusov
    • 1
    Email author
  • T. A. Smirnova
    • 1
    • 2
  • G. Ya. Kolomijtseva
    • 1
  1. 1.Belozersky Institute of Physico-Chemical BiologyLomonosov Moscow State UniversityMoscowRussia
  2. 2.Institute of Agricultural BiotechnologyMoscowRussia

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