Figure 9.

Effect of 3′-tail length on helicase activity and DNA binding. (A) Helicase activity. Reaction mixtures (10 μL) containing 20 mM Tris-HCl, pH 8.0, 5 mM MgCl₂, 50 nM duplex DNA substrate with 3′-T20, 3′-T15, 3′-T10, or 3′-T5 tails, 1 mM ATP, and 0, 25, 50, or 100 ng of UvrD2 were incubated for 5 min at 37 °C. The products were analyzed by native PAGE and visualized by autoradiography. A control reaction lacking enzyme that was heat denatured prior to PAGE is shown in lane Δ. The 3′-T20 substrate is shown at the bottom with the 5′-³²P label denoted by β. (B) DNA binding. Reaction mixtures (10 μL) containing 20 mM Tris-HCl, pH 8.0, 5 mM MgCl₂, 5% glycerol, 0.5 pmol of ³²P-labeled 3′-tailed DNAs as specified, and UvrD2 (200 ng) or 1 mM AMPPNP (where indicated by +) were incubated at 37 °C for 10 min. The mixtures were adjusted to 10% glycerol and then analyzed by native gel electrophoresis. The free DNAs and UvrD2-DNA complexes were visualized by autoradiography.