Figure 7.

Processivity of supercoil unwinding by wild-type vTopo and the K271E mutant using highly supercoiled MC^sp2. For the sake of clarity, only selected time points are shown in the gel figures. (A) Kinetic model for the disappearance of substrate into product and two intermediate pools (I1 and I2), followed by reversible cleavage and unwinding of intermediate pools to product. (B) Time course for unwinding using 5 nM MC^sp2 and 5 nM wild-type vTopo. Substrate (S), product (P), and intermediate bands (i^–2 to i^–7) were resolved by gel electrophoresis, visualized by SYBR Green staining, imaged, and quantified using the Gaussian curve fitting routine in QuantityOne. A representative plot of the Gaussian fits for the time point at 120 s is shown at the right. The time courses for the appearance and disappearance of substrate (green), products (black), and intermediate pools (blue and red) were fit by numerical integration and least-squares minimization to a stepwise supercoil unwinding model shown in panel A and the Supporting Information. (C) Time course for unwinding using 5 nM MC^sp2 and 5 nM K271E mutant. All steps in data acquisition and analysis are identical to those described in panel A.