FIGURE 3.

Gp32F fluorescence effects under conditions of helicase loading. Helicase loading assays were performed as described under “Experimental Procedures.” Buffer contained the following: 20 mm Tris acetate, pH 7.4, 10 mm magnesium acetate, 25 mm potassium acetate, and 1 mm DTT. The components of the helicase loading complex were added in the following order and preincubated: 100 nm Gp32F, 700 nm Oligo 1 (mixed sequence 70-mer), and 100 nm Gp59. Data show normalized fluorescence of this complex versus time after addition of 100 nm Gp41 and 1 mm ATP simultaneously. A, Gp32F fluorescence changes in the presence of ATP (red) or ATPγS (green) or in the absence of nucleotide cofactor (dark blue). B, controls using the same procedure as in A except in the absence of Gp41 (purple), Gp59 (orange), or ssDNA (light blue).