The coupled reaction can be monitored spectrophotometrically by m

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 Fludarabine concentration 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 GDC-0994 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

represent one standard deviation of the mean. Acknowledgements This work was supported by grants from Research Corporation for Science Advancement Adriamycin solubility dmso and from the University of Dayton Research Council to MEL, and by grants from the University of Dayton Graduate School to CF and BS. References 1. Cox MM, Goodman MF, Kreuzer KN, Sherratt DJ, Sandler SJ, Marians KJ: The importance of repairing stalled replication forks. Nature 2000,404(6773):37–41.PubMedCrossRef 2. Heller RC, Marians KJ: Replisome assembly and the direct restart ADAM7 of stalled replication forks. Nat Rev Mol Cell Biol 2006,7(12):932–943.PubMedCrossRef 3. Lee MS, Marians KJ: Escherichia coli replication factor Y, a component of the primosome, can act as a DNA helicase. Proc Natl Acad Sci USA 1987,84(23):8345–8349.PubMedCrossRef 4. Allen GC, Kornberg A: Assembly of the primosome of DNA replication in Escherichia coli. J Biol Chem 1993,268(26):19204–19209.PubMed 5. Liu J, Marians KJ: PriA-directed assembly of a primosome on D loop DNA. J Biol

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