Figure 2.

Determination of the sequence specificity of topoisomerase II on ribonucleotide-containing substrates. Topoisomerase II-mediated cleavage of substrates containing a ribonucleotide at different positions was performed as described in Materials and Methods. Cleavage products were isolated from a phenol/water interphase and analyzed by electrophoresis on a 12% denaturing polyacrylamide gel after proteinase K treatment. Lanes 1–8 and 10–17 show human topoisomerase IIα- and IIβ-mediated cleavage, respectively. Lanes 9 and 18 show DNA size markers (M) increasing in steps of two bases. The position of the ribonucleotide relative to the cleavage site is as indicated above the lanes. D represents the control DNA substrate. S indicates the cleavage substrate remaining in the interphase after phenol extraction. Cl indicates the cleavage product, for which migration is retarded by ∼1 base due to residual undigested protein. The asterisks indicate cleavage products having a longer protein fragment covalently linked due to partial proteinase K digestion (4,9).