Estimation of accessibility of DNA in chromatin from fluorescence measurements of electronic excitation energy transfer

Abstract

EUKARYOTIC DNA is complexed with proteins which influence its conformation, stability and function^1,2. Attempts to correlate the chemical and biological properties of chromatin have focused on the coverage of DNA by chromosomal proteins^3–8. As much as 50% of the DNA in chromatin is accessible to precipitants (such as basic dyes or polylysine)^3–6 or to limiting nucleolytic digestion^3,4,6–8, suggesting a model of segmental DNA accessibility in chromatin resembling a frayed telephone wire. Digestion extensively alters chromatin structure, however, and the results of experiments involving precipitation or digestion may be influenced by processes associated with the aggregation phenomena serving as experimental endpoints. We have used an alternative approach to characterise DNA accessibility in chromatin using the transfer of electronic excitation energy between pairs of fluorescent dyes bound to chromatin at low ratios of dye to phosphate. The results suggest that at least one-third of the DNA in chromatin retains a high affinity for basic dyes. The exclusion of dyes or other molecules⁹ from portions of chromatin may, however, depend on the conformation of chromatin as well as on the protein–DNA stoichiometry.