Figure 2.

Active site titration assay to measure the $K_{d(app)}^{DNA}$ for hPolε exo− binding to DNA. A preincubated solution of hPolε exo− (50 nm, UV concentration) and increasing concentrations of 5′-radiolabeled D-1 DNA substrate (10-125 nm) was rapidly mixed with dTTP (100 μm) and Mg²⁺ for 50 ms and then quenched with the addition of EDTA. All measurements were performed in triplicate and the average concentration of E•DNA complex that formed during the preincubation period, given by product concentration, was plotted against total D-1 DNA concentration and the data were fit to Equation 4 to yield a $K_{d(app)}^{DNA}$ of 22 ± 4 nm and an active enzyme concentration (E₀) of 14.7 ± 0.8 nm. Error bars represent the S.D. from the calculated average product concentration.