FIGURE 4.

FEN1 processing of substrates containing flaps of 30 nt under single-turnover conditions. A, representative time courses of FEN1 acting upon 30M (filled squares) and 30R (open squares). B, representative autoradiograms depicting cleavage of 30M (top panel) and 30R (bottom panel) under single-turnover conditions. The 0 reaction time is a negative control (see “Experimental Procedures”). Experimental conditions were 10 nm DNA substrate, 50 nm FEN1, 30 mm HEPES-KOH, 40 mm KCl, 8 mm MgCl₂, 5% (v/v) glycerol, 0.1 mg/ml BSA, pH 7.4. Reactions were performed at 37 °C using an RQF instrument and quenched with EDTA.