Typhoon 5 scanner

Nicking and religation assays were performed in 25 mM Hepes-NaOH, pH 7.0, 300 mM NaCl, 5 mM MgCl₂. Nicking assays contained 100 nM F-oriT57, religation assays (version 1) contained 100 nM F-oriT57 and 100 nM F-oriT20, and religation assays (version 2) contained 100 nM F-oriT35 and 100 nM non-fluorescent oriT57 (Fig 1B and Table S1). DNA oligomers were incubated with increasing concentrations of TraI in a shaking heatblock at 37°C and 300 rpm, for 1 h. The reactions were stopped by adding 2× stop solution (96% formamide, 20 mM EDTA, 0.1% bromophenol blue). The samples were boiled for 5 min at 95°C before loading on a denaturing gel (16% polyacrylamide, 7 M urea, 1× TBE). The gel was run at 50 V for 15 min followed by 100 V for 1 h at room temperature and then imaged on an Amersham Typhoon 5 scanner with excitation at 488 nm, using the Cy2 emission filter (515–535 nm).

Labelling of newly synthesized mitochondrially expressed proteins was performed as previously described⁴² . Briefly, DdCBE-treated cells at approximately 80% confluency were incubated in methionine/cysteine-free medium for 10 min. The medium was then replaced with methionine/cysteine-free medium containing 10% dialysed FCS and emetine dihydrochloride (100 μg ml⁻¹) to inhibit cytosolic translation. Following a 20 min incubation, 120 μCi ml⁻¹ of (³⁵S)-methionine was added and the cells were incubated for 60 min. After washing with 1X PBS, cells were lysed, and 30 μg of protein was loaded on either 16% Tris- glycine or 16% Tricine SDS–PAGE gels, as indicated in the figure. Dried gels were visualized with a PhosphorImager system (Amersham Typhoon 5 scanner). Densitometric quantification was performed using ImageJ along the midpoint of each lane and plotted using GraphPad Prism.

Fluorescently labeled DNA (50 nM, Table S1) was incubated for 15 min with 0–1,600 nM of protein (see the Results section) in 25 mM Hepes-NaOH, pH 7.0, 300 mM NaCl at room temperature before adding 6× native loading dye (3× TBE, 30% glycerol, 0.125% bromophenol blue). The samples were resolved on a native gel (5% polyacrylamide, 0.75× TBE) in 0.75× TBE running buffer at 50 V for 90 min at 4°C. Gels were imaged on an Amersham Typhoon 5 scanner with excitation at 488 nm, using the Cy2 emission filter (515–535 nm).