Stepwise Nucleosome Translocation by RSC Remodeling Complexes

Abstract

The SWI/SNF family of chromatin-remodeling enzymes are important regulators of chromatin structure in transcriptional activation and in the creation and maintenance of nucleosome free regions. These enzymes regulate DNA accessibility by using the free energy from ATP hydrolysis to reposition and restructure nucleosomes, but the mechanism by which these enzymes move DNA around the histone octamer remains unclear. In this work, we study RSC, a prototypical SWI/SNF-family enzyme by using single-molecule FRET to monitor in real time the structural dynamics of individual nucleosomes during remodeling. By employing a variety of labeling schemes, we were able to observe transient intermediates during remodeling and assess the relative contributions of DNA translocation, DNA unwrapping, and octamer conformational change to the nucleosome remodeling process. We found that RSC primarily slides DNA around the nucleosome without large-amplitude lifting of the DNA from the nucleosomal surface or substantial displacement of the H2A-H2B dimer. We also observe that RSC-mediated translocation of DNA across the nucleosome occurs in a stepwise manner and that the translocation step size is 1-2 bp at both sites where the DNA enters and exits the nucleosome. Our results suggest that the motion of DNA around the nucleosome is directly coupled to DNA translocation by the remodeler's ATPase domain at its internal nucleosome binding site, and that other domains of the catalytic subunit or the accessory subunits do not play an active role in translocating DNA around the nucleosome.