Relaxation of chromatin structure by ethidium bromide binding: determined by viscometry and histone dissociation studies.

Abstract

The effects of ethidium bromide intercalation on chromatin structure were monitored by viscometry and analysis of histone dissociation. Investigation of the NaCl concentration dependence of chromatin viscosity showed that the reduced viscosity (etared) was very low up to 0.4 M NaCl and increased gradually when the salt concentration was raised further. In chromatin intercalated by ethidium bromide, etared was not significantly different at low salt concentrations (up to 0.2 M NaCl). However, when the salt concentration was raised further, the viscosity response curve increased sharply to reach viscosities about 4-5 times higher than those for nonintercalated chromatin. The increase in viscosity was proportional to the increase in fluorescence intensity, when the ratio of ethidium bromide to DNA mucleotide was raised. The transition of intercalated chromatin into the relaxed form was reversible, dependent on the nature of the electrolyte and cooperative, as indicated by the small increase in salt concentration required to obtain chromatin relaxation. Investigation of the NaCl concentration dependence of histone dissociation revealed that total histones and each individual histone fraction were released from intercalated chromatin at much reduced NaCl concentrations. The midpoints of the dissociation curves of the individual histones ranged from 0.30 to 0.45 M NaCl and fell within the same range where the drastic viscosity change occurred. These results indicate that intercalation of ethidium bromide labilizes chromatin structure to relaxation by moderately elevated salt concentrations. It is suggested that the labilization is caused by changes in the DNA helix conformation due to dye intercalation decreasing the stability of histone-DNA interactions.