Circular dichroism microscopy of compact forms of DNA and chromatin in vivo and in vitro: cholesteric liquid-crystalline phases of DNA and single dinoflagellate nuclei.
Two highly condensed structures of DNA have been analyzed in the circular dichroism (CD) microscope: the cholesteric liquid-crystalline phase of DNA and the nucleus of a dinoflagellate (Prorocentrum micans). In both cases, the DNA shows a helical cholesteric organization, but the helical pitch equals about 2500 nm in the first case and 250 nm in the second one. Since the absorption band of DNA is located at 260 nm, the reflection and absorption bands are well separated in the cholesteric phase of DNA and are overlapping in the dinoflagellate nucleus. However, both structures give a very strong negative CD signal at 265 nm. We show that this very strong signal cannot correspond to a Borrmann effect, i.e., to a superposition of the absorption and reflection bands, but is a differential absorption of left versus right circularly polarized light. This anomalous differential absorption is probably due to a significant scattering of light, inside of the structure, which produces a resonance phenomenon in the absorption band of the chromophore. Therefore, for any helical structure containing a chromophore, the apparent CD can be expressed as CD = [(epsilon L - epsilon R)cl] + (psi L - psi R) + (SL - SR) The first term is true absorption and is located in the absorption band of the chromophore, and the last term is true scattering and is observed at the wavelength corresponding to the helical pitch of the structure. The second term (psi L - psi R) corresponds to the anomalous differential absorption observed in dense superhelical structures of DNA. It superimposes to the first term in the absorption band of the chromophore. psi L - psi R is a measure of the perfection of the helical structure and of the density of chromophores in the material. Intercalative dyes [ethidium bromide and meso-tetrakis(4-N-methylpyridyl)porphine (H2TMpyP-4) and its nickel(II) derivative (NiIITMpyP-4)] were inserted in the dinoflagellate chromatin. The CD signal recorded in their absorption band mimics the signal observed in the absorption band of DNA. In both structures, the negative sign of the CD at 265 nm indicates that the twist occurring between DNA. In both structures, the negative sign of the CD at 265 nm indicates that the twist occurring between DNA molecules is left-handed, and we show that this situation is the most frequently encountered in vivo and vitro.
Livolant
F
F
Centre de Biologie Cellulaire, CNRS, Ivry-sur-Seine, France.
Maestre
M F
MF
D000818:Animals; D002467:Cell Nucleus / Q000032:analysis*; D002843:Chromatin / Q000032:analysis*; D002942:Circular Dichroism; D004247:DNA / Q000528:radiation effects; D004141:Dinoflagellida; D004996:Ethidium; D009690:Nucleic Acid Conformation; D014466:Ultraviolet Rays
D002843:Chromatin; D004247:DNA; D004996:Ethidium