Remodeling of Individual Nucleosomes in Nucleosome Arrays

Adenosin triphosphate (ATP)-dependent nucleosome remodeling factors sculpt the nucleosomal landscape of eukaryotic chromatin. They deposit, evict, or reposition nucleosomes along DNA in a process termed nucleosome sliding. Remodeling has traditionally been analyzed using mononucleosomes as a model substrate. In vivo, however, nucleosomes form extended arrays with regular spacing. Here we describe how regularly spaced nucleosome arrays can be reconstituted in vitro and how these arrays can be used to dissect remodeling in the test tube. We outline two assays. Both assays exploit the changes in the accessibility of DNA to restriction enzymes during the remodeling reaction. The first assay uses the restriction enzyme to cleave the restriction site as soon as it becomes accessible during remodeling. As such, this assay mostly reports the kinetic parameter of the "forward" reaction of nucleosome remodeling. In contrast, the second assay measures how fast a particular nucleosome in the array reaches its steady-state position.