Restriction enzyme digestion in the presence or absence of RuvCGFP or RDG

The DNA substrate was a HJ containing the Eco RI recognition sequence in one of the arms. Digestion was performed on the free junction or after binding by RuvCGFP or RDG. Each reaction mixture contained 2 pmol of the junction and 2.5 U of Eco RI (New England Biolabs). Prebinding of the junction by RuvCGFP or RDG was carried out in the digestion buffer (supplied by New England Biolabs) for 10 min on ice before enzyme addition. The molar ratio of the junction to RuvCGFP or RDG was 1:10. Immediately following enzyme addition, the reaction mixtures were transferred to a 37°C water bath. Reactions were stopped by the addition of 0.2% SDS, and samples were analyzed by electrophoresis in 3% agarose gel at room temperature (10 V cm⁻¹). DNA was visualized by ethidium bromide staining. Quantification in plots shows means ± SEM from three independent experiments.