ETHIDIUM-BROMIDE AS A PROBE OF CONFORMATIONAL HETEROGENEITY OF DNA IN CHROMATIN - ROLE OF HISTONE H-1

The accessibility and the tertiary structure of the DNA inside chromatin [from calf thymus] were studied by using ethidium bromide (EB) as a fluorescent probe. The exclusion model of binding was refined by introducing a parameter .alpha. (0 < .alpha. < 1) which measures the accessibility of the DNA and by taking into account when necessary the existence of 2 sets of binding sites. Predicted and experimental isotherms were correlated. EB binding to native or partially histone depleted chromatin under various conditions was completely described. In native chromatin 95% of the DNA (.alpha. = 0.95) appears to be accessible to EB but 2 sets of sites are present. The first one corresponds to .alpha. = 0.13 and is characterized by an affinity constant which is higher by 2 orders of magnitude than that relative to pure DNA. The second set corresponds to .alpha. = 0.82 and the corresponding binding constant is only 3 or 4 times lower than that of pure DNA. The sites with high affinity are still present after treatment with formalydehyde but disappear after removal of histone H1. By comparison with chromatin treated with deoxycholate or with artificial complexes between H1 and DNA, high affinity sites were found only when all of the histones are bound to DNA. An .alpha. value around 0.8 is still obtained in 1 M NaCl treated chromatin, pointing to the fact that histones H3 and H4 are preventing 20% of the DNA to intercalate EB.