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. 2000 May;78(5):2614–2627. doi: 10.1016/S0006-3495(00)76806-8

Restrained torsional dynamics of nuclear DNA in living proliferative mammalian cells.

M Tramier 1, K Kemnitz 1, C Durieux 1, J Coppey 1, P Denjean 1, R B Pansu 1, M Coppey-Moisan 1
PMCID: PMC1300851  PMID: 10777758

Abstract

Physical parameters, describing the state of chromatinized DNA in living mammalian cells, were revealed by in situ fluorescence dynamic properties of ethidium in its free and intercalated states. The lifetimes and anisotropy decays of this cationic chromophore were measured within the nuclear domain, by using the ultra-sensitive time-correlated single-photon counting technique, confocal microscopy, and ultra-low probe concentrations. We found that, in living cells: 1) free ethidium molecules equilibrate between extracellular milieu and nucleus, demonstrating that the cation is naturally transported into the nucleus; 2) the intercalation of ethidium into chromatinized DNA is strongly inhibited, with relaxation of the inhibition after mild (digitonin) cell treatment; 3) intercalation sites are likely to be located in chromatin DNA; and 4) the fluorescence anisotropy relaxation of intercalated molecules is very slow. The combination of fluorescence kinetic and fluorescence anisotropy dynamics indicates that the torsional dynamics of nuclear DNA is highly restrained in living cells.

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Selected References

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  1. Ambroz M., MacRobert A. J., Morgan J., Rumbles G., Foley M. S., Phillips D. Time-resolved fluorescence spectroscopy and intracellular imaging of disulphonated aluminium phthalocyanine. J Photochem Photobiol B. 1994 Feb;22(2):105–117. doi: 10.1016/1011-1344(93)06955-3. [DOI] [PubMed] [Google Scholar]
  2. Angerer L. M., Moudrianakis E. N. Interaction of ethidium bromide with whole and selectively deproteinized deoxynucleoproteins from calf thymus. J Mol Biol. 1972 Feb 14;63(3):505–521. doi: 10.1016/0022-2836(72)90444-5. [DOI] [PubMed] [Google Scholar]
  3. Arents G., Moudrianakis E. N. Topography of the histone octamer surface: repeating structural motifs utilized in the docking of nucleosomal DNA. Proc Natl Acad Sci U S A. 1993 Nov 15;90(22):10489–10493. doi: 10.1073/pnas.90.22.10489. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Ashraf S. I., Ip Y. T. Transcriptional control: repression by local chromatin modification. Curr Biol. 1998 Sep 24;8(19):R683–R686. doi: 10.1016/s0960-9822(98)70435-x. [DOI] [PubMed] [Google Scholar]
  5. Axelrod D. Carbocyanine dye orientation in red cell membrane studied by microscopic fluorescence polarization. Biophys J. 1979 Jun;26(3):557–573. doi: 10.1016/S0006-3495(79)85271-6. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Axelrod D. Fluorescence polarization microscopy. Methods Cell Biol. 1989;30:333–352. [PubMed] [Google Scholar]
  7. Baker T. A., Bell S. P. Polymerases and the replisome: machines within machines. Cell. 1998 Feb 6;92(3):295–305. doi: 10.1016/s0092-8674(00)80923-x. [DOI] [PubMed] [Google Scholar]
  8. Bastiaens P. I., Squire A. Fluorescence lifetime imaging microscopy: spatial resolution of biochemical processes in the cell. Trends Cell Biol. 1999 Feb;9(2):48–52. doi: 10.1016/s0962-8924(98)01410-x. [DOI] [PubMed] [Google Scholar]
  9. Brochon J. C. Maximum entropy method of data analysis in time-resolved spectroscopy. Methods Enzymol. 1994;240:262–311. doi: 10.1016/s0076-6879(94)40052-0. [DOI] [PubMed] [Google Scholar]
  10. Carey M. The enhanceosome and transcriptional synergy. Cell. 1998 Jan 9;92(1):5–8. doi: 10.1016/s0092-8674(00)80893-4. [DOI] [PubMed] [Google Scholar]
  11. Clendenning J. B., Naimushin A. N., Fujimoto B. S., Stewart D. W., Schurr J. M. Effect of ethidium binding and superhelix density on the supercoiling free energy and torsion and bending constants of p30 delta DNA. Biophys Chem. 1994 Nov;52(3):191–218. doi: 10.1016/0301-4622(94)00038-l. [DOI] [PubMed] [Google Scholar]
  12. Coppey-Moisan M., Brunet A. C., Morais R., Coppey J. Dynamical change of mitochondrial DNA induced in the living cell by perturbing the electrochemical gradient. Biophys J. 1996 Nov;71(5):2319–2328. doi: 10.1016/S0006-3495(96)79472-9. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Coppey-Moisan M., Delic J., Magdelenat H., Coppey J. Principle of digital imaging microscopy. Methods Mol Biol. 1994;33:359–393. doi: 10.1385/0-89603-280-9:359. [DOI] [PubMed] [Google Scholar]
  14. Dayel M. J., Hom E. F., Verkman A. S. Diffusion of green fluorescent protein in the aqueous-phase lumen of endoplasmic reticulum. Biophys J. 1999 May;76(5):2843–2851. doi: 10.1016/S0006-3495(99)77438-2. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Delic J., Coppey J., Magdelenat H., Coppey-Moisan M. Impossibility of acridine orange intercalation in nuclear DNA of the living cell. Exp Cell Res. 1991 May;194(1):147–153. doi: 10.1016/0014-4827(91)90144-j. [DOI] [PubMed] [Google Scholar]
  16. Durieux C., Brunet A. C., Geeraert V., Coppey J., Coppey-Moisan M. A transient decrease of electrochemical gradient stabilizes DNA structural change in single mitochondria of living cells. Biol Cell. 1999 Nov;91(8):597–604. [PubMed] [Google Scholar]
  17. Echols H. Multiple DNA-protein interactions governing high-precision DNA transactions. Science. 1986 Sep 5;233(4768):1050–1056. doi: 10.1126/science.2943018. [DOI] [PubMed] [Google Scholar]
  18. Favard C., Pager J., Locker D., Vigny P. Incorporation of ethidium bromide in the Drosophila salivary gland approached by microspectrofluorometry: evidence for the presence of both free and bound dye in the nuclei of cells in viable conditions. Eur Biophys J. 1997;25(4):225–237. doi: 10.1007/s002490050035. [DOI] [PubMed] [Google Scholar]
  19. Gao M., Knipe D. M. Genetic evidence for multiple nuclear functions of the herpes simplex virus ICP8 DNA-binding protein. J Virol. 1989 Dec;63(12):5258–5267. doi: 10.1128/jvi.63.12.5258-5267.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Grosschedl R. Higher-order nucleoprotein complexes in transcription: analogies with site-specific recombination. Curr Opin Cell Biol. 1995 Jun;7(3):362–370. doi: 10.1016/0955-0674(95)80091-3. [DOI] [PubMed] [Google Scholar]
  21. Guest C. R., Hochstrasser R. A., Dupuy C. G., Allen D. J., Benkovic S. J., Millar D. P. Interaction of DNA with the Klenow fragment of DNA polymerase I studied by time-resolved fluorescence spectroscopy. Biochemistry. 1991 Sep 10;30(36):8759–8770. doi: 10.1021/bi00100a007. [DOI] [PubMed] [Google Scholar]
  22. Hayashi J., Takemitsu M., Goto Y., Nonaka I. Human mitochondria and mitochondrial genome function as a single dynamic cellular unit. J Cell Biol. 1994 Apr;125(1):43–50. doi: 10.1083/jcb.125.1.43. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Hazlett T. L., Johnson A. E., Jameson D. M. Time-resolved fluorescence studies on the ternary complex formed between bacterial elongation factor Tu, guanosine 5'-triphosphate, and phenylalanyl-tRNAPhe. Biochemistry. 1989 May 2;28(9):4109–4117. doi: 10.1021/bi00435a073. [DOI] [PubMed] [Google Scholar]
  24. Heath P. J., Clendenning J. B., Fujimoto B. S., Schurr J. M. Effect of bending strain on the torsion elastic constant of DNA. J Mol Biol. 1996 Aug 2;260(5):718–730. doi: 10.1006/jmbi.1996.0432. [DOI] [PubMed] [Google Scholar]
  25. Hernández L. I., Zhong M., Courtney S. H., Marky L. A., Kallenbach N. R. Equilibrium analysis of ethidium binding to DNA containing base mismatches and branches. Biochemistry. 1994 Nov 8;33(44):13140–13146. doi: 10.1021/bi00248a025. [DOI] [PubMed] [Google Scholar]
  26. Hogan M. E., Rooney T. F., Austin R. H. Evidence for kinks in DNA folding in the nucleosome. Nature. 1987 Aug 6;328(6130):554–557. doi: 10.1038/328554a0. [DOI] [PubMed] [Google Scholar]
  27. Härd T., Kearns D. R. Reduced DNA flexibility in complexes with a type II DNA binding protein. Biochemistry. 1990 Jan 30;29(4):959–965. doi: 10.1021/bi00456a017. [DOI] [PubMed] [Google Scholar]
  28. Jupe E. R., Sinden R. R., Cartwright I. L. Stably maintained microdomain of localized unrestrained supercoiling at a Drosophila heat shock gene locus. EMBO J. 1993 Mar;12(3):1067–1075. doi: 10.1002/j.1460-2075.1993.tb05748.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Knutson J. R., Davenport L., Brand L. Anisotropy decay associated fluorescence spectra and analysis of rotational heterogeneity. 1. Theory and applications. Biochemistry. 1986 Apr 8;25(7):1805–1810. doi: 10.1021/bi00355a053. [DOI] [PubMed] [Google Scholar]
  30. Lakowicz J. R., Szmacinski H., Nowaczyk K., Berndt K. W., Johnson M. Fluorescence lifetime imaging. Anal Biochem. 1992 May 1;202(2):316–330. doi: 10.1016/0003-2697(92)90112-k. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Lamond A. I., Earnshaw W. C. Structure and function in the nucleus. Science. 1998 Apr 24;280(5363):547–553. doi: 10.1126/science.280.5363.547. [DOI] [PubMed] [Google Scholar]
  32. Leuba S. H., Bustamante C., Zlatanova J., van Holde K. Contributions of linker histones and histone H3 to chromatin structure: scanning force microscopy studies on trypsinized fibers. Biophys J. 1998 Jun;74(6):2823–2829. doi: 10.1016/S0006-3495(98)77989-5. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Luger K., Mäder A. W., Richmond R. K., Sargent D. F., Richmond T. J. Crystal structure of the nucleosome core particle at 2.8 A resolution. Nature. 1997 Sep 18;389(6648):251–260. doi: 10.1038/38444. [DOI] [PubMed] [Google Scholar]
  34. Macgregor R. B., Jr, Clegg R. M., Jovin T. M. Viscosity dependence of ethidium-DNA intercalation kinetics. Biochemistry. 1987 Jun 30;26(13):4008–4016. doi: 10.1021/bi00387a040. [DOI] [PubMed] [Google Scholar]
  35. Manders E. M., Kimura H., Cook P. R. Direct imaging of DNA in living cells reveals the dynamics of chromosome formation. J Cell Biol. 1999 Mar 8;144(5):813–821. doi: 10.1083/jcb.144.5.813. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. McMurray C. T., van Holde K. E. Binding of ethidium to the nucleosome core particle. 1. Binding and dissociation reactions. Biochemistry. 1991 Jun 11;30(23):5631–5643. doi: 10.1021/bi00237a001. [DOI] [PubMed] [Google Scholar]
  37. Meyer-Almes F. J., Porschke D. Mechanism of intercalation into the DNA double helix by ethidium. Biochemistry. 1993 Apr 27;32(16):4246–4253. doi: 10.1021/bi00067a012. [DOI] [PubMed] [Google Scholar]
  38. Olmsted J., 3rd, Kearns D. R. Mechanism of ethidium bromide fluorescence enhancement on binding to nucleic acids. Biochemistry. 1977 Aug 9;16(16):3647–3654. doi: 10.1021/bi00635a022. [DOI] [PubMed] [Google Scholar]
  39. Owen-Hughes T., Workman J. L. Experimental analysis of chromatin function in transcription control. Crit Rev Eukaryot Gene Expr. 1994;4(4):403–441. [PubMed] [Google Scholar]
  40. Pulleyblank D. E., Morgan A. R. The sense of naturally occurring superhelices and the unwinding angle of intercalated ethidium. J Mol Biol. 1975 Jan 5;91(1):1–13. doi: 10.1016/0022-2836(75)90368-x. [DOI] [PubMed] [Google Scholar]
  41. Rydberg B., Holley W. R., Mian I. S., Chatterjee A. Chromatin conformation in living cells: support for a zig-zag model of the 30 nm chromatin fiber. J Mol Biol. 1998 Nov 20;284(1):71–84. doi: 10.1006/jmbi.1998.2150. [DOI] [PubMed] [Google Scholar]
  42. Schröter H., Maier G., Ponstingl H., Nordheim A. DNA intercalators induce specific release of HMG 14, HMG 17 and other DNA-binding proteins from chicken erythrocyte chromatin. EMBO J. 1985 Dec 30;4(13B):3867–3872. doi: 10.1002/j.1460-2075.1985.tb04159.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Schurr J. M., Schurr R. L. DNA motions in the nucleosome core particle: a reanalysis. Biopolymers. 1985 Oct;24(10):1931–1940. doi: 10.1002/bip.360241007. [DOI] [PubMed] [Google Scholar]
  44. Selvin P. R., Scalettar B. A., Langmore J. P., Axelrod D., Klein M. P., Hearst J. E. A polarized photobleaching study of chromatin reorientation in intact nuclei. J Mol Biol. 1990 Aug 20;214(4):911–922. doi: 10.1016/0022-2836(90)90345-M. [DOI] [PubMed] [Google Scholar]
  45. Smith S. B., Finzi L., Bustamante C. Direct mechanical measurements of the elasticity of single DNA molecules by using magnetic beads. Science. 1992 Nov 13;258(5085):1122–1126. doi: 10.1126/science.1439819. [DOI] [PubMed] [Google Scholar]
  46. Sobell H. M., Tsai C. C., Gilbert S. G., Jain S. C., Sakore T. D. Organization of DNA in chromatin. Proc Natl Acad Sci U S A. 1976 Sep;73(9):3068–3072. doi: 10.1073/pnas.73.9.3068. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Sogo J. M., Ness P. J., Widmer R. M., Parish R. W., Koller T. Psoralen-crosslinking of DNA as a probe for the structure of active nucleolar chromatin. J Mol Biol. 1984 Oct 5;178(4):897–919. doi: 10.1016/0022-2836(84)90318-8. [DOI] [PubMed] [Google Scholar]
  48. Studitsky V. M., Kassavetis G. A., Geiduschek E. P., Felsenfeld G. Mechanism of transcription through the nucleosome by eukaryotic RNA polymerase. Science. 1997 Dec 12;278(5345):1960–1963. doi: 10.1126/science.278.5345.1960. [DOI] [PubMed] [Google Scholar]
  49. Thomas J. C., Allison S. A., Appellof C. J., Schurr J. M. Torison dynamics and depolarization of fluorescence of linear macromolecules. II. Fluorescence polarization anisotropy measurements on a clean viral phi 29 DNA. Biophys Chem. 1980 Oct;12(2):177–188. doi: 10.1016/0301-4622(80)80050-0. [DOI] [PubMed] [Google Scholar]
  50. Verkman A. S., Armijo M., Fushimi K. Construction and evaluation of a frequency-domain epifluorescence microscope for lifetime and anisotropy decay measurements in subcellular domains. Biophys Chem. 1991 Apr;40(1):117–125. doi: 10.1016/0301-4622(91)85036-p. [DOI] [PubMed] [Google Scholar]
  51. Wahl P., Paoletti J., Le Pecq J. B. Decay of fluorescence emission anisotropy of the ethidium bromide-DNA complex. Evidence for an internal motion in DNA. Proc Natl Acad Sci U S A. 1970 Feb;65(2):417–421. doi: 10.1073/pnas.65.2.417. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Wang J. C. The degree of unwinding of the DNA helix by ethidium. I. Titration of twisted PM2 DNA molecules in alkaline cesium chloride density gradients. J Mol Biol. 1974 Nov 15;89(4):783–801. doi: 10.1016/0022-2836(74)90053-9. [DOI] [PubMed] [Google Scholar]
  53. Widom J. Structure, dynamics, and function of chromatin in vitro. Annu Rev Biophys Biomol Struct. 1998;27:285–327. doi: 10.1146/annurev.biophys.27.1.285. [DOI] [PubMed] [Google Scholar]
  54. Winzeler E. A., Small E. W. Fluorescence anisotropy decay of ethidium bound to nucleosome core particles. 2. The torsional motion of the DNA is highly constrained and sensitive to pH. Biochemistry. 1991 May 28;30(21):5304–5313. doi: 10.1021/bi00235a025. [DOI] [PubMed] [Google Scholar]
  55. Wu P. G., Fujimoto B. S., Song L., Schurr J. M. Effect of ethidium on the torsion constants of linear and supercoiled DNAs. Biophys Chem. 1991 Dec;41(3):217–236. doi: 10.1016/0301-4622(91)85038-r. [DOI] [PubMed] [Google Scholar]
  56. Yao J., Lowary P. T., Widom J. Twist constraints on linker DNA in the 30-nm chromatin fiber: implications for nucleosome phasing. Proc Natl Acad Sci U S A. 1993 Oct 15;90(20):9364–9368. doi: 10.1073/pnas.90.20.9364. [DOI] [PMC free article] [PubMed] [Google Scholar]

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