Anisotropy-Based Nucleosome Repositioning Assay

Abstract

Most eukaryotic DNA is tightly packaged into nucleosomes that render these sequences largely inaccessible for transcription or repair. Molecular motors called chromatin remodelers use an ATP-dependent mechanism to relieve the inhibition of these processes by sliding or disassembling the nucleosomes. This allows them to serve an essential role in the regulation of gene expression and genomic integrity. The sliding of nucleosomes along DNA can be studied directly by monitoring the associated changes in the fluorescence anisotropy of fluorophores attached to the ends of the DNA. Nucleosome repositioning can also be monitored indirectly through the ATP hydrolysis of the chromatin remodeler during the sliding reaction. Here we discuss how the kinetic data collected in these experiments can be analyzed by simultaneous global nonlinear least squares (NLLS) analysis using simple sequential "n-step" mechanisms to obtain estimates of the macroscopic rate of nucleosome repositioning and of the stoichiometry of coupling ATP binding and hydrolysis to this reaction.