Quantitative TLC-based ATPase Assay

Thin layer chromatography (TLC)-based ATPase assays were performed using [α-³²P]ATP (Hartmann Analytic)^{36 (link),37 (link)}. To quantify DNA-stimulated ATPase activity, 0.5 µM protein or protein complex were combined with 1 mM of a 43-nucleotide ssDNA (5’-GGCCGCGAGCCGGAAATTTAATTATAAACCAGACCGTCTCCTC-3’). 0.5 µM protein or protein complex or equivalent protein–DNA mixtures were incubated with 1 mM [α-³²P]ATP in 50 mM HEPES-NaOH, pH 7.5, 80 mM NaCl, 5 mM MgCl₂, 2 mM DTT at 30 °C for up to 60 min. 5 µl of sample were withdrawn at selected time points and reactions were quenched with 5 µl of 100 mM EDTA. 0.8 µl of the samples were spotted on a PEI-cellulose TLC plate and chromatographed with 1 M acetic acid, 0.5 M LiCl, 20 % (v/v) ethanol. The corresponding ADP and ATP spots were visualized using a Storm 860 phosphorimager (GMI, USA) and quantified using ImageQuant software (version 5.2; Cytiva). Data were plotted and analyzed using Prism (version 9.0; GraphPad), the ATPase activity was calculated as the number of hydrolyzed ATP molecules per protein molecule per minute, by fitting quantified data to the equation V = (A_fast * V_fast² + A_slow * V_slow²)/(A_fast * V_fast + A_slow * V_slow); A_fast/slow, amplitudes of the fast/slow hydrolysis phases; V_fast/slow, rates of the fast/slow hydrolysis phases [min^-1]; V, ATP hydrolyzed as a function of time [min⁻¹].