FIGURE 3.

Properties of the AdnAB helicase. A, reaction mixtures (10 μl) containing 20 mm Tris-HCl (pH 8.0), 0.9 mm DTT, 0.1 μm (1 pmol) 5′ ³²P-labeled 24-bp blunt duplex DNA, 0.85 pmol of nuclease-dead AdnAB, and 2 mm MgCl₂, 2 mm ATP, or 2 mm AMPPNP (where specified by +) were incubated for 15 min at 37 °C. B, ATP titration. Reaction mixtures (10 μl) containing 20 mm Tris-HCl (pH 8.0), 2 mm MgCl₂, 0.9 mm DTT, 0.1 μm (1 pmol) 5′ ³²P-labeled 24-bp blunt duplex DNA, 0.85 pmol of nuclease-dead AdnAB, and increasing amounts of ATP as specified were incubated for 15 min at 37 °C. C, nucleotide specificity. Reaction mixtures (10 μl) containing 20 mm Tris-HCl (pH 8.0), 2 mm MgCl₂, 0.9 mm DTT, 0.1 μm (1 pmol) 5′ ³²P-labeled 24-bp blunt duplex DNA, 0.85 of pmol nuclease-dead AdnAB, and either no nucleotide (− lane) or 1 mm of the indicated NTP or dNTP were incubated for 15 min at 37 °C. Enzyme was omitted from the control reaction in lane -E. D, divalent cation specificity. Reaction mixtures containing 20 mm Tris-HCl (pH 8.0), 2 mm ATP, 0.1 μm (1 pmol) 5′ ³²P-labeled 24-bp blunt duplex DNA, 0.85 pmol of nuclease-dead AdnAB, and 2 mm of the specified divalent cation were incubated for 15 min at 37 °C. The reaction products were analyzed by native PAGE and visualized by autoradiography. DNA-containing reaction mixtures lacking AdnAB that were heat-denatured prior to PAGE are included in lanes marked by ▵.