Figure 1.

(A) Escherichia coli topoisomerase I relaxes negatively supercoiled DNA more efficiently than E. coli topoisomerase III in bulk experiments. The gel shows relaxation of negatively supercoiled DNA by either Topo I or Topo III. Identical mass amounts of protein and DNA were incubated for the same period of time and the resulting relaxed DNA products were analyzed in an ethidium bromide-stained agarose gel. The experiment shows that Topo I relaxes DNA more fully than comparable amounts of Topo III. The amount of protein used is shown at the top of the gel as well as the equivalent molarity for each lane. An amount of 200 ng (0.72 fmol) of DNA was used in each lane. R marks the position of relaxed DNA and SC the position of supercoiled DNA. (B) Substrates used for single-molecule experiments. Three different types of DNA molecules were employed for the single-molecule experiments: intact dsDNA (top), DNA with a single-stranded bubble or bulge (middle), and DNA with a mismatched region (bottom). The bulge and mismatch regions were used in experiments with positively supercoiled DNA as type IA topoisomerase require single-stranded regions for activity. The intact DNA molecules were used for experiments with negatively supercoiled DNA.