FIGURE 4.

EMSA experiments with 39-mer dsDNA and various Topo-44 active site mutants. 4 μm DNA was incubated with 10 μm protein at 65 °C for 30 min, and the products were separated on a 6% native polyacrylamide gel. The wild-type protein and some of the Glu-215 mutants form clear protein-DNA complexes or a cleaved product. The His-200 mutants show a faint gel shift, demonstrating an effect on DNA binding by the mutations. The Lys-218, Arg-131, and Y226F mutants show no visible shift. The gel was cropped to remove a failed assay.