Oligonucleotides were suspended in 10 mM Tris·HCl pH 8, 50 mM NaCl, 1 mM EDTA at a 2:1 molar ratio of non-labeled DNA to fluorescein-labeled DNA. The DNAs were incubated at 95°C for 5 min, MM-102 nmr slow-cooled to 70°C and incubated at that temperature for 60 min, and slow-cooled to 25°C. Duplex DNAs were gel-purified through 6% polyacrylamide gels using 100 mM Tris borate pH 8.3, 2 mM EDTA as the electrophoresis buffer. The DNAs were excised from the polyacrylamide gels, electroeluted using the same electrophoresis

buffer, dialyzed against 10 mM Tris·HCl pH 8, 5 mM MgCl2, aliquoted, and stored at -20°C. Equilibrium DNA binding assays Fluorescence polarization spectroscopy was performed at 25°C with a Beacon 2000 fluorescence polarization system (Invitrogen). Serial dilutions of PriA or PriB were made into 20 mM Tris·HCl {Selleck Anti-cancer Compound Library|Selleck Anticancer Compound Library|Selleck Anti-cancer Compound Library|Selleck Anticancer Compound Library|Selleckchem Anti-cancer Compound Library|Selleckchem Anticancer Compound Library|Selleckchem Anti-cancer Compound Library|Selleckchem Anticancer Compound Library|Anti-cancer Compound Library|Anticancer Compound Library|Anti-cancer Compound Library|Anticancer Compound Library|Anti-cancer Compound Library|Anticancer Compound Library|Anti-cancer Compound Library|Anticancer Compound Library|Anti-cancer Compound Library|Anticancer Compound Library|Anti-cancer Compound Library|Anticancer Compound Library|Anti-cancer Compound Library|Anticancer Compound Library|Anti-cancer Compound Library|Anticancer Compound Library|Anti-cancer Compound Library|Anticancer Compound Library|buy Anti-cancer Compound Library|Anti-cancer Compound Library ic50|Anti-cancer Compound Library price|Anti-cancer Compound Library cost|Anti-cancer Compound Library solubility dmso|Anti-cancer Compound Library purchase|Anti-cancer Compound Library manufacturer|Anti-cancer Compound Library research buy|Anti-cancer Compound Library order|Anti-cancer Compound Library mouse|Anti-cancer Compound Library chemical structure|Anti-cancer Compound Library mw|Anti-cancer Compound Library molecular weight|Anti-cancer Compound Library datasheet|Anti-cancer Compound Library supplier|Anti-cancer Compound Library in vitro|Anti-cancer Compound Library cell line|Anti-cancer Compound Library concentration|Anti-cancer Compound Library nmr|Anti-cancer Compound Library in vivo|Anti-cancer Compound Library clinical trial|Anti-cancer Compound Library cell assay|Anti-cancer Compound Library screening|Anti-cancer Compound Library high throughput|buy Anticancer Compound Library|Anticancer Compound Library ic50|Anticancer Compound Library price|Anticancer Compound Library cost|Anticancer Compound Library solubility dmso|Anticancer Compound Library purchase|Anticancer Compound Library manufacturer|Anticancer Compound Library research buy|Anticancer Compound Library order|Anticancer Compound Library chemical structure|Anticancer Compound Library datasheet|Anticancer Compound Library supplier|Anticancer Compound Library in vitro|Anticancer Compound Library cell line|Anticancer Compound Library concentration|Anticancer Compound Library clinical trial|Anticancer Compound Library cell assay|Anticancer Compound Library screening|Anticancer Compound Library high throughput|Anti-cancer Compound high throughput screening| pH 8, 10% (v/v) glycerol, 50 mM NaCl, 1 mM 2-mercaptoethanol, 0.1 mg/ml bovine serum albumin (BSA) and incubated

with 1 nM fluorescein-labeled DNA. Apparent dissociation constants (Kd,app) were calculated by determining the concentration of either PriA or PriB required to bind 50% of the fluorescein-labeled DNA (Curve Expert 1.3). The unbound state is reported by the fluorescence anisotropy of the fluorescein-labeled DNA in the presence of buffer alone. The fully-bound state is reported by the fluorescence anisotropy of the fluorescein-labeled DNA in the presence of

a sufficient concentration of PriA or PriB to saturate the fluorescence anisotropy signal. Data are reported in triplicate and associated uncertainties represent one standard deviation of the mean. DNA unwinding assays DNA substrates were diluted to 1 nM in 20 mM Tris·HCl pH 8, 50 mM NaCl, 3 mM MgCl2, 1 mM 2-mercaptoethanol, 1 mM ATP. For unwinding assays involving PriB proteins, indicated concentrations of wild type PriB or PriB:K34A were added to the DNA and incubated for 5 min Racecadotril on ice. Indicated concentrations of PriA were added to the reaction mixtures and incubated at 37°C for 10 min to facilitate duplex DNA unwinding. Reactions were stopped by addition of SDS to a final concentration of 1%. The amount of duplex DNA unwound was determined by measuring the fluorescence anisotropy of the samples Etomoxir in vivo following addition of SDS. Fluorescence anisotropy values were compared to the fluorescence anisotropy of the DNA substrate incubated in buffer alone (fully intact DNA substrate) and the fluorescence anisotropy of the DNA substrate after being heated to 95°C and rapidly cooled to 25°C (fully denatured DNA substrate) to calculate the fraction of DNA unwound. Data are reported in triplicate and associated uncertainties represent one standard deviation of the mean. ATP hydrolysis assays PriA-catalyzed ATP hydrolysis was measured using a coupled spectrophotometric assay that has been previously described [32].