Ethidium bromide as a probe of conformational heterogeneity of DNA in chromatin. The role of histone H1.

The accessibility and the tertiary structure of the DNA inside chromatin were studied by using ethidium bromide (EB) as a fluorescent probe. The exclusion model of binding was refined by introductina a parameter alpha (0less than alpha less than 1) which measures the accessibility of the DNA and by taking into account when necessary the existence of two sets of binding sites. We were thus able to fit predicted and experimental isotherms and then to describe completely EB binding to native or partially histone depleted chromatin under various conditions. Itn native chromatin 95% of the DNA (alpha = 0.95) appears to be accessible to EB but two sets of sites are present. The first one corresponds to alpha = 0.13 and is characterized by an affinity constant which is higher by two orders of magnitude than that relative to pure DNA. The second set corresponds to alpha = 0.82 and the corresponding binding constant is only three or four times lower than that of pure DNA. The sites with high affinity are still present after treatment with formaldehyde but disappear after removal of histon H1. By comparison with chromatin treated with deoxycholate of with artifical complexes between H1 and DNA, high affinity sites were found only when all of the histons are bound to DNA. An alpha value around 0.8 is still obtained in 1 M NaC1 treated chromatin, pointing to the fact that histones H3 and H4 are preventing 20% of the DNA to intercalate EB.