ATP Hydrolysis Assay for DDX5

In vitro ATP hydrolysis assays were performed using an enzyme-coupled absorbance assay (36 (link)). ATP hydrolysis rate was measured using 400 nM purified, recombinant MBP-DDX5-GST or mutants in the presence and absence of 250 ug/ml total yeast RNA (Sigma). k_obs values were calculated using the formula: V₀ = (A₃₄₀/min × 2.5)/(6.22 × 10⁻³μM), where k_obs(min⁻¹) = V₀/protein concentration. DDX5 fused to an N-terminal MBP and C-terminal GST tag (MBP-DDX5-GST) was expressed in E.coli and purified by affinity chromatography. DDX5 mutants were purified by the same method. N=3, error bars represent standard deviation.