Histone Exchange Assay for SWR Chromatin Remodeling

The histone exchange assay was conducted using a protocol modified from (32 (link)). Each reaction is 25 μl and is composed of three parts. Part A, which constitutes 60% of the reaction volume, contains 4 nM purified SWR in 25 mM HEPES–KOH (pH 7.6), 0.5 mM EGTA, 0.1 mM EDTA, 5 mM MgCl₂, 0.17 μg/μl BSA, 50 mM NaCl, 10% glycerol, 0.02% NP-40. Part B, which constitutes 20% of the reaction volume, contains 75 nM Cy3-labeled AA nucleosome and 550 nM H2A.Z–H2B^FL dimers in 10 mM Tris–HCl (pH 7.5), 1 mM EDTA, 50 mM NaCl. Part C is 1 mM ATP and represents 20% of the reaction volume. Part A and Part B were mixed together before Part C was added to initiate the reaction. The reaction was left at room temperature (∼22°C) for the indicated times before they were quenched by the addition of 62.5 ng of lambda phage DNA (New England Biolabs). Five microliter of Nap1 at 3.5 μM in buffer S [70% (w/v) sucrose, 10 mM Tris–HCl (pH 7.8), 1 mM EDTA] was added to the reaction immediately before resolving the nucleosomes on 6% polyacrylamide/0.5× TBE gels. In-gel Cy3 fluorescence was detected by a Typhoon 9500 scanner (GE Healthcare) and densitometry was performed using the ImageQuant software. For the Swc5 rescue experiments, Swc5 and/or H2A–H2B dimer were added to the Part A and B mixture at the indicated final concentrations before adding in Part C.