Influence of Linker DNA on Nucleosome Structure according to Single-Particle Fluorescence Microscopy Data

Abstract: Mononucleosomes assembled using DNA templates of various lengths that contain a nucleosome-positioning sequence are widely used in molecular biological studies, but their structural features require detailed investigation. The single-particle fluorescence microscopy based on the Förster resonance energy transfer was used to compare the structure of nucleosomes with two 20 bp length DNA linkers and core-nucleosomes without linkers (CN) in solutions containing 150 mM KCl and 5 mM MgCl₂ (or without MgCl₂) as well as in solutions with increased ionic strength (0.5 and 0.7 M KCl). It was found that these nucleosomes are present in solutions as two dominant subpopulations, which differ in the DNA folding on the histone octamer. It was revealed that CN and 2LN differ in the ratio of these subpopulations, and the differences increase in the presence of Mg²⁺ ions. With an increase in the ionic strength, conformational reorganizations occur in the core region of the nucleosomes. The character of the reorganizations differs in 2LN and CN at 0.5 M KCl but becomes similar at 0.7 M KCl. The obtained data indicate that, despite the similar conformations of 2LN and CN, the same factors can induce a different effect on the structure of these nucleosomes, and it should be taken into account when, for example, studying the interactions of mononucleosomes with various nuclear proteins in vitro.