Figure 3.

Nucleotide binding assays. Formation of ternary Enzyme-DNA-dNTP complexes with T4-exo⁻ (A) and L412M-exo⁻ (B) were measured as a function of dTTP concentration using the chain-terminated DNA substrate illustrated in Figure 1A. 10 mM MgCl₂ was present. dTTP binding quenches 2AP fluorescence in a concentration-dependent manner (•), but dFTP does not at the concentrations tested (□); however, dFTP at 50 (○) and 150 μM (▼) reduced the ability of dTTP to quench 2AP fluorescence. Equilibrium dissociation constants (K_ds) for dTTP binding were determined by fitting the data (solid line) to the hyperbolic equation, q = [Q] [dTTP]/K_d + [dTTP], where q is the observed fluorescence quench at various dTTP concentrations and Q is the maximum fluorescence quench observed at saturating dTTP. K_is for dFTP were determined in dTTP binding reactions with either 50 or 150 μM dFTP; dFTP was a competitive inhibitor of dTTP binding as demonstrated by the Lineweaver-Burk plots (insets). K_d values for dTTP binding and K_i values for dFTP for T4-exo-⁻ and L412M-exo⁻ are given below the quench curves.