FIGURE 6.

Modulation of topo IIβ reaction by SP120-RNA: Time course. A, tag-purified FLAG-topo IIβ (0.04 pmol) was preincubated with increasing amounts of FLAG-SP120 that contained endogenous RNA (SP120-RNA) or corresponding amounts of RNA purified from FLAG-SP120 (0.02, 0.04, 0.08, 0.16, and 0.32 pmol in lanes 1–5, respectively). Relaxation was started by addition of supercoiled DNA (pUC18) and the reaction products were separated by 1% agarose gel electrophoresis. S, substrate; E, enzyme alone. The lagging ladder bands are relaxed topoisomers. The reaction is totally dependent on the presence of ATP (not shown). B, two hypothetical modes of relaxation time course under restricted dose of enzyme. Note that a portion of substrate molecules remains unrelaxed when the reaction is completely processive, whereas all the substrate is converted eventually to relaxed forms in the distributive mode. C, relaxation of pUC18 by topo IIβ alone (0.01 pmol) or by topo IIβ-SP120-RNA equimolar complex (0.01 pmol each) was stopped at the times indicated. I, supercoiled DNA. D, unrelaxed DNA bands in C were quantified by densitometry and plotted in values relative to time 0.