Figure 5.

Kinetic scheme for the FEN1 reaction. FEN1 binds and bends a short DF substrate at diffusion-limited kinetics and commits to cleavage within the first encounter. After cleavage, the 5′ flap is released instantaneously while nicked product release occurs in two steps. FEN1 can rebind/rebend the nicked product at diffusion-limited kinetics, albeit with a lower on-rate than the substrate. With longer flaps, FEN1 can miss cleavage and dissociate from the bent substrate, requiring more than one attempt at cleavage. k_{bending-internal} and product release rates are shown for non-EQ DF-6,1_Internal at 40 mM KCl in presence of Mg²⁺. k_STO at 40 mM KCl is shown for EQ DF-6,1_Flap in Supplementary Figure S2C; k_STO is independent of KCl concentration and it has been shown to be similar for both EQ DF-6,1_Flap and non-EQ DF-6,1_Flap (8). The substrate and product on/off rates are determined in presence of Ca²⁺; substrate EQ DF-6,1_Internal at 100 mM KCl (Figure 2B) and product at 40 mM KCl (Supplementary Figure S2B). Based on data with the product (Supplementary Figure S2B), we do not anticipate a significant change in k_on-bending of the substrate with lower KCl concentration.