Fig. 6.

AdnB Trp325-Arg326 mutation uncouples ATP hydrolysis from ssDNA translocation. Reaction mixtures (10 μL) containing 20 mM Tris⋅HCl (pH 8.0), 2 mM MgCl₂, 1 mM DTT, 0.1 μM 5′ ³²P-labeled ssDNA substrate (shown at Bottom), 1 mM ATP, and 0.1 pmol wild-type or mutant AdnAB (where indicated by +) were incubated for 20 min at 37 °C. The reactions were quenched by adjustment of the mixtures to 45% formamide and 25 mM EDTA. The products were analyzed by electrophoresis through a 15-cm 18% polyacrylamide gel containing 7 M urea in 45 mM Tris-borate and 1.2 mM EDTA and then visualized by autoradiography. The sites of cleavage of the 24-mer ssDNA by the AdnA and AdnB nuclease domains are indicated by arrows at Bottom.