Ls 6000sc scintillation counter

Native PAGE running buffer (25 mM Tris, 192 mM glycine, pH 8.3 from Bio-Rad) was degassed overnight in an anaerobic chamber with vigorous stirring. The duplex character of substrates was assessed by an annealing titration (see Supporting Experimental Section), and completely annealed duplexes were used for EMSAs and in activity assays (see below). Radioactivity was detected using an LS 6000SC Scintillation Counter (Beckman). In an anaerobic chamber, 10 nM to 2 μM UvrC (by cluster) was incubated for 30 min at room temperature with 100 nM dsDNA in activity buffer or UvrC buffer. DNA protein mixtures were electrophoresed at 50 V for 2 h at room temperature using Mini-PROTEAN TGX 4–20% Precast Gels (Bio-Rad). Gels were exposed on a phosporimaging screen as described in the Supporting Experimental Section. For each replicate, bands were quantified using Image Lab (Bio-Rad) and the fraction of bound DNA was plotted as a function of free UvrC concentration and fit to the Hill function using Origin (OriginLab Corporation). The apparent dissociation constant reported is the free UvrC concentration when half of the DNA substrates are bound. From three independent trials, the apparent dissociation constants were averaged over and are reported with the standard error of the mean.

To measure uptake driven by TF0F1, 25 µl proteoliposomes containing 2–3.5 µg VGLUT were diluted into 775 µl uptake buffer (150 mM K gluconate, 5 mM HEPES-KOH pH 7.3, 5 mM Mg gluconate, 0.2 mM glutamate, 10 µCi/ml L-[3,4-³H]-glutamic acid (PerkinElmer) and 2 mM MgATP), for a final Cl⁻ concentration of 5 mM. To measure uptake in the absence of TF0F1 (VGLUT only), the uptake buffer at pH 7.3 contained 150 mM K gluconate, 10 mM HEPES-KOH pH 7.3 and 5 mM Mg gluconate (KGluc pH 7.3). At pH 6.0, the buffers contained 0.2 mM glutamate and 10 µCi/ml L-[3,4-³H]-glutamic acid with either 150 mM K gluconate, 10 mM MES-KOH pH 6.0 and 5 mM Mg gluconate (KGluc pH 6) or 160 mM KCl, 10 mM MES-KOH pH 6.0 and 5 mM MgCl₂ (KCl pH 6). The reactions were incubated at 30° C for the times indicated and stopped by rapid filtration through 0.45 µm HAWP discs (Millipore). The filters were washed 3 times with cold wash buffer (150 mM K gluconate, 5 mM HEPES-KOH, pH 7.3, 5 mM Mg gluconate, dried, solubilized in CytoScint (MP Biomedicals) and the bound radioactivity measured using an LS 6000SC scintillation counter (Beckman). All experiments were performed in duplicate or triplicate and repeated at least three times. Glutamate uptake was normalized to the mass of reconstituted VGLUT added to each reaction.