Figure 5: Activity of Topo I mutants against different substrates.
(a) Crystal structure of the Topo I ssDNA covalent complex (Protein Data Bank ID: 3PX7)43. The four domains (D I–IV) of the enzyme are shown in different colours, with the two mutated residues in mutant study, R173 and Y319, highlighted in red. (b,c) Investigating the ssDNA- and ssRNA-unknotting activity of WT Topo I and two mutants using ssDNA knot, dTKj (b) and ssRNA knot, TKj (c) as probes. Probe substrates were 80 nM, treated by 320 nM of proteins (30 min incubation at 37 °C). (d) Topological relaxations of dTKj (80 nM) catalysed by increasing concentrations of Topo I R173A mutant (30 min incubation at 37 °C). The enzyme in lanes 1–5 was 10, 20, 40, 80 and 160 nM. (e) Summary of activities of the WT Topo I and two mutants tested in this work against different substrates. The sc plasmid relaxation activities were taken from previous work44.
