FIGURE 7.

Slow binding of A257T to the replication fork. A, a mixture of WT or A257T gp4 (200 nm), T7 DNAP (200 nm), dTTP (4 mm), and radiolabeled ds40 (20%) replication fork substrate was incubated for various time intervals (Δt: 5 s to 30 min). A solution of MgCl₂ and dT90 trap was then added to initiate the unwinding reaction, which was quenched in 5 s. The DNA substrate and the unwound ssDNA were resolved by native-PAGE. B, the image shows the extent of unwinding in 5 s in reactions containing T7 DNAP alone or T7 DNAP plus A257T or WT gp4 as a function of Δt. C, fraction of replication fork unwound as a function of Δt. The data were fit to single exponential equation to obtain the rate of helicase assembly in the presence of T7 DNAP: WT (filled circles) at 0.03 s⁻¹ and A257T (open circles) at 0.001 s⁻¹.