Histones were assembled and purified using the salt gradient dialysis method as previously described44 (link). Briefly, Xenopus laevis core histones were expressed in BL21 Rosetta cells and purified from inclusion bodies. Histone octamers were reconstituted and purified by gel filtration chromatography. To determine the optimal ratio for nucleosome assembly, histone octamers were mixed with varying concentrations of 601 nucleosomal DNA for test assemblies. Nucleosomes were assembled according to the optimal DNA:histone ratio and purified by gel filtration chromatography.
Native gel-shift assays were performed in 20 mM HEPES pH 7.5, 50 mM NaCl, 5% glycerol, 1 mM DTT, 0.1 mg/mL BSA, 1 mM MgCl2 (link), and 1 mM ADP (or AMPPNP). The concentration of nucleosomes or free DNA was held constant at 50 μM, and Sth1 constructs were added at a range of concentrations to a maximum of 10 μM. The reactions were incubated for 30 min at room temperature, and 10 μL aliquots were loaded onto a 4–20% TBE gel (Bio-Rad) in 0.5 × TBE. The gels were run at 4 °C and 150 V for 3 h (nucleosome) or 45 min (free DNA), and then stained with SYBR Gold (GE Healthcare) and imaged on a Bio-Rad Gel Doc Imager. The bands corresponding to the nucleosome core particle or the 21-bp DNA duplex were quantified densitometrically using the program ImageJ. For each lane, the nucleosome band was quantified and normalized to the no-Sth1 control, and these values were subtracted from 1 to obtain the fraction of nucleosomes bound.
Native gel-shift assays were performed in 20 mM HEPES pH 7.5, 50 mM NaCl, 5% glycerol, 1 mM DTT, 0.1 mg/mL BSA, 1 mM MgCl2 (link), and 1 mM ADP (or AMPPNP). The concentration of nucleosomes or free DNA was held constant at 50 μM, and Sth1 constructs were added at a range of concentrations to a maximum of 10 μM. The reactions were incubated for 30 min at room temperature, and 10 μL aliquots were loaded onto a 4–20% TBE gel (Bio-Rad) in 0.5 × TBE. The gels were run at 4 °C and 150 V for 3 h (nucleosome) or 45 min (free DNA), and then stained with SYBR Gold (GE Healthcare) and imaged on a Bio-Rad Gel Doc Imager. The bands corresponding to the nucleosome core particle or the 21-bp DNA duplex were quantified densitometrically using the program ImageJ. For each lane, the nucleosome band was quantified and normalized to the no-Sth1 control, and these values were subtracted from 1 to obtain the fraction of nucleosomes bound.
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