Mapping CENP-A Nucleosome Structure

CENP-A nucleosomes assembled with HEX-labelled 147 bp α-satellite DNA20 (link) were reconstituted and then purified using a sucrose gradient. An amount of 4 μg of HEX-labelled CENP-A nucleosomes alone or complexed to CENP-N^NT were used in each reaction. The hydroxyl radical cleavage reaction was initiated by addition of 5 μl of 40 mM FeAmSO₄/80 mM EDTA, 2 M ascorbate and 2.4% H₂O₂ to a 30 μl reaction mixture. Each reaction was carried out for 5 min at room temperature, and terminated with 200 μl of stop solution (0.1% SDS, 25 mM EDTA, 1% glycerol and 100 mM Tris, pH 7.4). Further phenol/chloroform extraction and ethanol precipitation was carried out to extract DNA fragments. Samples were separated by denaturing PAGE (10% polyacrylamide, 7 M urea, 88 mM Tris-borate and 2 mM EDTA, pH 8.3)20 (link). Gels were imaged on a Typhoon 9200 imager (GE Healthcare). Band intensities were quantified from ImageJ from three independent experiments.