Asymmetric Nucleosome Remodeling Assay

We prepared hexasomes according to published methods (30 (link),31 (link)). Hexamers were reconstituted, containing either H2A/H2B dimers or biotin-modified H2A/H2B S112C dimers, then converted to full nucleosomes with modified or native dimers, respectively, to generate asymmetrically modified nucleosomes. Converted nucleosomes were purified by sucrose gradients. Remodeling reactions were carried out as described above, in the presence or absence of streptavidin, and reactions terminated by the addition of 200 ng plasmid DNA. Reactions were digested with 10 u HindIII for 60 min at 37°C then loaded directly onto 5% acrylamide nucleoprotein gels and the wet gels imaged by fluorography (GE Amersham Typhoon). The amount of products before and after HindIII digestion was quantified by densitometric analysis of the fluorographs and the fraction remaining uncut by HindIII calculated and plotted.