Unraveling Nucleosome Dynamics Using Fluorescence Fluctuation Analysis

Abstract

Nucleosomes are the smallest repeating units of eukaryotic chromatin. Target DNA sites for various biological processes are often buried inside these nucleosomes. There are various mechanisms by which DNA accessibility is increased. Recent studies show that DNA spontaneously and transiently unwraps from the histone core, presenting itself to the protein machineries. We have studied this dynamics using fluorescence correlation analysis. Our approach is to use a FRET construct where the acceptor (Cy5) is on the histone while the donor (Cy3) is moved along the length of the DNA, starting from the 5′ terminus all the way to the center of the dyad axis. We have used a combination of experimental FCS techniques and Monte Carlo simulations to determine the timescales of the nucleosome wrapping-unwrapping process. Conventional FCS methods face the challenge of separating the kinetic contributions from the diffusion contributions to the autocorrelation functions. Here, we will present and discuss a variety of approaches aimed to overcome these difficulties, including the analysis of the donor-acceptor cross-correlation and correlation of the generalized polarization function.