DNA cleavage assay

A total of 1.5 μg of PUC19 plasmid DNA was incubated in 50 mM Tris-HCl 18 mM NaCl, pH 7.2 buffer with either 0.5 μM complex 1 or DMSO in a total reaction mixture of 125 μl. Reaction mixtures were then exposed to light as described above. The control reactions without exposure to light for DMSO and 0.5 μM complex 1 were set up in parallel. All reactions were then incubated overnight at 37 °C. 5 μl of 0.05% bromophenol blue loading buffer was added to 25 μl of each reaction mixture prior to electrophoresis on a 1.2% agarose gel run at 100 V for 1 hour. Gels were stained with 1 μg/ml Ethidium Bromide (EtBr) and then visualised on a UGenius fluorescence gel imager (Syngene, Cambridge, UK). DNA bands were quantified using GelQuantNET software (Biochem lab solutions, San Francisco, Ca, USA). The ratio of nicked DNA versus supercoiled DNA was determined and then the fold change in that ratio between no light conditions and light conditions at each dosage was calculated. To determine DNA cleavage capacity of complex 1 following intercalation of EtBr, 19 μM of PUC19 plasmid DNA base pairs was saturated with 13.1 μM EtBr for 5 minutes prior to addition of 0.5 μM complex 1. To determine DNA cleavage capacity of complex 1 under hypoxic conditions PUC19 plasmid DNA was incubated in an environment containing <0.1% oxygen in a hypoxic chamber (Don Whitley VA500 anaerobic workstation fitted with individually gassed humidified boxes (Don Whitley Scientific, Shipley, West Yorkshire) for 30 min in the dark prior to light treatment.