Figure 5.

Topoisomerase II-mediated cleavage of an RNA/DNA hybrid. The RNA/DNA hybrid (R/D) or the control DNA substrate (D) labelled at the 5′-end of the top strand was incubated with human topoisomerase II as described in Materials and Methods. After ethanol precipitation the samples were submitted to electrophoresis on a 12% denaturing polyacrylamide gel and cleavage complex formation was visualized upon autoradiography. The left and right panels show human topoisomerase IIα- and IIβ-mediated cleavage, respectively. Lanes 1 and 2 (both panels) show experiments performed with the control DNA substrate in the absence and presence of topoisomerase II as indicated. Lanes 3 and 4 (both panels) represent cleavage reactions performed with the RNA/DNA hybrid in the absence and presence of topoisomerase II, as indicated. M represents DNA size markers increasing in steps of two bases. S_R/D and S_D indicate the positions of the hybrid and control DNA substrates having a labelled 25mer RNA and DNA strand, respectively. Cl_D represents the position of cleavage products obtained with the control DNA substrate. Low levels of substrate degradation are seen on the gel, which are most pronounced for the RNA strand due to the presence of residual RNase activity.