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. Author manuscript; available in PMC: 2009 Sep 9.
Published in final edited form as: Biochemistry. 2008 Aug 15;47(36):9355–9364. doi: 10.1021/bi800725q

Figure 6.

Figure 6

The CxxC motifs are not required for helicase activity. (A) Aliquots (5 μg) of recombinant wild-type (WT) UvrD2 and mutants C607A-C610A and C625A-C628A were analyzed by SDS-PAGE. The Coomassie blue-stained gel is shown. The sizes (kDa) and positions of marker proteins are indicated on the left. (B) ATPase. Reaction mixtures (10 μL) containing 20 mM Tris-HCl, pH 8.0, 1 mM [γ-32P]ATP, 2 mM MgCl2, 50 ng of salmon sperm DNA, and wild-type or mutant UvrD2 as specified were incubated for 5 min at 37 °C. (C) Helicase. Reaction mixtures (10 μL) containing 5 mM MgCl2, 50 nM 3′-tailed duplex DNA substrate, 1 mM ATP, and 10, 20, or 40 ng of wild-type or mutant UvrD2 as specified were incubated for 5 min at 37 °C. The products were analyzed by native PAGE and visualized by autoradiography. A control reaction without added protein is included in lane -E; a control reaction lacking enzyme that was heat denatured prior to PAGE is shown in lane Δ.