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. 2014 Jul 17;42(14):9005–9020. doi: 10.1093/nar/gku640

Figure 6.

Figure 6.

Effects of RNA on the relaxation activity of topo IIβ domain-deletion mutants. (A) Domain structure of deletion mutants used here that are Flag-tagged on N-terminus (not shown in the figure). (B) Inhibitory effects of total RNA on the relaxation activity. Tag-purified topo IIβ and the domain mutants (20 fmol) were incubated with 5 ng of supercoiled pUC18 DNA. Increasing amounts of RNA (5, 50, 500 ng) were added to the reaction. Positions of supercoiled form (I) and relaxed form (Ir) DNA are indicated on the right. (C) Fractionation of relaxation intermediates into enzyme-bound and released DNA by using 80 fmol immobilized topo IIβ domain mutants (on-bead assay). After incubation with 5 ng of supercoiled DNA as described in ‘Methods’ section, product DNA was fractionated into ‘bound’ and ‘unbound’ by magnetic separation. (D) Effects of reaction temperature on the retention of relaxation intermediates on the enzyme. DNA in unbound fraction indicates that the supercoiled substrate remained almost unreacted at 0°C, but no unreacted substrate remained at 15°C. Difference in the ladder position between 15 and 37°C is due to the temperature-dependent alteration of the writhe for relaxed form (Ir). Results for the WT enzyme are shown in Figure 3C.