Unless otherwise stated, all measurements were carried out in SF50 buffer at 25°C. Stopped-flow experiments were carried out in KinTek SF-2004 and BioLogic SFM 300 apparatuses. Quenched-flow experiments were performed in a KinTek RQF-3 instrument. Post-mixing concentrations are stated in all experiments. In experiments requiring nucleotide-free RecQ, nucleotide contamination was removed by pre-incubation with 0.02-U/ml apyrase for 15 min at 25°C. Pi liberation from ATP was followed using a fluorescently labeled Pi binding protein (MDCC-PBP) (12 (link)). MDCC-PBP calibration was performed as described earlier (13 (link),14 (link)). mdATP and mdADP were excited at 280 nm and fluorescence emission was detected through a 420-nm long-pass filter utilizing FRET (Förster Resonance Energy Transfer) from aromatic residues of RecQ.
Steady-state ATPase measurements were carried out in SF50 buffer plus 50-μg/ml bovine serum albumin using a pyruvate kinase-lactate dehydrogenase (PK-LDH) linked assay (14-U/ml PK, 20-U/ml LDH, 1-mM ATP, 1-mM phosphoenol pyruvate, 200-μM NADH (nicotinamide adenine dinucleotide, reduced form)). Time courses of NADH absorbance (ϵ340 nm = 6220 M−1cm−1) were followed in a Shimadzu UV-2101PC spectrophotometer.
Steady-state ATPase measurements were carried out in SF50 buffer plus 50-μg/ml bovine serum albumin using a pyruvate kinase-lactate dehydrogenase (PK-LDH) linked assay (14-U/ml PK, 20-U/ml LDH, 1-mM ATP, 1-mM phosphoenol pyruvate, 200-μM NADH (nicotinamide adenine dinucleotide, reduced form)). Time courses of NADH absorbance (ϵ340 nm = 6220 M−1cm−1) were followed in a Shimadzu UV-2101PC spectrophotometer.
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