Fluorescence-based ATPase Activity Assay

ATPase activity was measured as described previously (Ahel et al., 2009 (link)) by monitoring the fluorescence intensity of a phosphate binding protein (Brune et al., 1994 (link), Ferreira et al., 2007 (link)) labeled with a coumarin-based fluorescent dye, 7-diethylamino-3-((((2-maleimidyl)ethyl)amino)carbonyl) coumarin (MDCC), as a readout of the amount of inorganic phosphate (P_i) generated by ATP hydrolysis. Upon binding P_i, the fluorescence of the labeled phosphate binding protein (MDCC-PBP) increases and its emission wavelength shifts. The increase in fluorescence was recorded in solution using a Spark 10 M (Tecan) instrument. 25 μL ATPase reactions were carried out in 50 mM Tris pH 7.9, 50 mM NaCl, and 5 mM MgCl₂ buffer with 80 nM ALC1 and 1 mM ATP. Where indicated, 50 μM NAD⁺ and 80 nM PARP1 (Thermo Fisher) and/or 120 nM 239 bp dsDNA were added. Upon incubation for 2 h at 37°C, phosphate sensor (Thermo Fisher) was added at a final concentration of 0.5 μM and immediately measured (excitation = 420 nm, emission = 465 nm).