The structure of chromatin: interaction of ethidium bromide with native and denatured chromatin

Abstract

The binding of ethidium bromide, as monitored by fluorescence enhancement, to chromatin [WI-L2 human lymphoblastoid cells] prepared by nuclease [micrococcal] digestion was compared with the binding of the dye to sheared chromatin. The nuclease preparation (native chromatin) is characterized by a high affinity region of the Scatchard plot (r [r = concentration of bound ethidium bromide/concentration of DNA] = 0-0.025, K1 [affinity constant for binding] = 1 .times. 106 M-1), a transition (r = 0.025-0.05), and a low affinity region (r = 0.05-0.12, K2 = 3 .times. 105 M-1). The final amount of ethidium bromide bound per base is 0.12 as compared wiht 0.20 for free DNA. Sheared chromatin has the 2 regions of high and low affinity (K1 = 2 .times. 106 M-1, K2 = 2 .times. 105 M-1), but the transition is much reduced or absent. Binding of the dye to native chromatin is independent of salt at concentrations ranging from 0.2 mM EDTA to 10 mM Tris-Cl, 10 mM NaCl, 0.2 mM EDTA, while sheared chromatin and DNA both bind ethidium bromide electrostatically and by intercalation at the low salt concentration, leading to extensive energy transfer. The phosphate groups in native chromatin are unavailable to external cations even at very low salt. Polarization of fluorescence of ethidium bromide intercalated into native chromatin at low r is very high, indicating a highly rigid structure. As r approaches 0.02, there is a very rapid depolarization; at r = 0.03, the polarization is no greater than that of the dye intercalated into DNA. Depolarization is not due to energy transfer. The Scatchard plot derived for the bulk preparation of native chromatin is very similar to the one derived for the monomer .nu. body. These results indicate that the DNA in native chromatin is in a very rigid form, with its phosphate anions neutralized by structural components, not by free salt. Ethidium bromide intercalation appears partially to disrupt this structure, perhaps by unwinding, leading to slight changes in its properties.