Phosho enol pyruvic acid

ATPase activity was measured using an ATP-regenerating system as previously reported (33 (link)). The ATPase assay were carried out at 23°C in a buffer contained 10 mM Tris-HCl (pH8.0), 100 mM KCl, 2 mM MgCl₂, 0.2 mM NADH Roche 10107735001), 200 μg/ml pyruvate kinase from rabbit muscle (Roche 10128155001), 2.5 mM Phosho(enol) pyruvic acid (Sigma P7252). First, 1 mM ATP was added to the solution, followed by different concentrations of DNA and finally 100 nM RHAU helicase to start the ATPase assay. The absorbance at 340 nm was measured using a Tecan Infinite F200 plate reader.

ATP (A2383), AMP-PNP (A2647), ADP (A2754) were purchased from Sigma-Aldrich. The ATP regenerating system [200 μg/ml pyruvate kinase from rabbit muscle (Roche 10128155001), 2.5 mM Phosho(enol) pyruvic acid (Sigma P7252)] was mixed with ATP before use of ATP. The ADP (Sigma A2754) solution was treated with hexokinase (Sigma H4502) and D-glucose (1st BASE) to remove the contaminating ATP as described previously (34 (link)). The ADP solution (80 mM) was incubated with 200 mM D-glucose, 2 mM MgCl₂, 0.04 U/μl hexokinase (Sigma H4502-500UN) in room temperature for 2 h. After incubation, the enzyme was eliminated from these solution using a Amicon Ultra-0.5 column. The ADP·AlF was prepared by incubating 10 mM ATP-depleted ADP, 50 mM NaF (Sigma 67414) and 10 mM AlCl₃ (Sigma 563919) as described previously (35 (link)).