FIGURE 2.

The rate of RecBCD translocation along ssDNA in the 5′ to 3′ direction is faster than in the 3′ to 5′ direction, and the rates are coupled asymmetrically. Stopped-flow experiments were performed by mixing a preformed complex of RecBCD (37.5 nm) and DNA (50 nm) with 5 mm ATP, 10 mm MgCl₂, and 7.5 mg/ml heparin (concentrations after mixing) in buffer M₂₅₀ at 25 °C. The lengths of the twin ssDNA extensions, L, are indicated. All insets show the linear dependence of lag time on ssDNA extension length L. A, monitoring 3′ to 5′ ssDNA translocation of RecBCD using DNA I. Inset, rate = 1409 ± 109 nt/s. B, monitoring 5′ to 3′ ssDNA translocation of RecBCD using DNA II. Inset, rate = 1922 ± 72 nt/s. C, monitoring 3′ to 5′ ssDNA translocation of RecBCD^K177Q using DNA I. Inset, rate = 1289 ± 14 nt/s. D, monitoring 5′ to 3′ ssDNA translocation of RecB^K29QCD using DNA II. Inset, rate = 1162 ± 11 nt/s. E, [ATP] dependence of the 3′ to 5′ translocation rates of RecBCD^K177Q (red) and WT RecBCD (black). For WT RecBCD, K_m = 337 ± 30 μm and V_max = 1515 ± 33 nt/s; for RecBCD^K177Q, K_m = 327 ± 14 μm and V_max = 1377 ± 14 nt/s. F, [ATP] dependence of the 5′ to 3′ translocation rates of RecB^K29QCD (green) and WT RecBCD (black). For WT RecBCD, K_m = 420 ± 89 μm and V_max = 2037 ± 109 nt/s; for RecB^K29QCD, K_m = 534 ± 84 μm and V_max = 1325 ± 56 nt/s. AU, arbitrary units.