The coupled reaction can be monitored spectrophotometrically by measuring the decrease in absorbance at 340 nm due to NADH oxidation. Primosome proteins at indicated concentrations were incubated with indicated concentrations of DNA and ATP in 20 mM Hepes pH 8, 50 mM NaCl, 7 mM 2-mercaptoethanol, 2 mM phosphoenol pyruvate, 0.1 mM NADH,

14 units/ml pyruvate kinase, 20 units/ml lactate dehydrogenase, 0.1 mg/ml BSA at 25°C. Steady-state Δ[NADH]/Δt rates were calculated using the molar extinction coefficient 6,220 M-1·cm-1 for NADH, and these rates are equivalent to Δ[ATP]/Δt. The kinetic parameters K m and k cat were determined with respect to DNA and with respect SHP099 mouse to ATP by fitting the ATP hydrolysis rates to the Michaelis-Menten equation, where S = either DNA or ATP (Curve Expert 1.3). Values of k cat were determined by dividing V max by the concentration of PriA in the reaction. Data are reported in triplicate and associated uncertainties

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