Figure 4.

Examination of the effect of multiple ribonucleotides on topoisomerase II-mediated cleavage. (A) Two ribonucleotides located one in each strand do not exert an additive effect on cleavage. Substrates harbouring a ribonucleotide in either one or both strands were incubated with human topoisomerase IIα as described in Materials and Methods and the SDS-stopped samples were analyzed on an 8% SDS–polyacrylamide gel. Cleavage levels were determined after PhosphorImager scanning of the gel and are schematically presented relative to the cleavage level obtained with the control DNA substrate (D). As indicated below the histogram, one set of substrates contained either a ribonucleotide at the +1 position of the top strand (+1t), of the bottom strand (+1b) or of both strands (+1t,+1b). The other set included substrates having a ribonucleotide at the –2 position of the top strand (–2t), of the bottom strand (–2b) or of both strands (–2t,–2b). Data represent the averages of three independent experiments and standard deviations are indicated by error bars. (B) Ribonucleotides present in the same strand do not exert an additive effect on cleavage. Cleavage experiments were performed as in (A). Cleavage levels were determined after PhosphorImager scanning of the gel and are schematically presented in the histogram relative to the cleavage level obtained with the control DNA substrate (D). The substrates used in the experiment contained a row of four consecutive ribonucleotides in the top strand either at positions –4 to –1 or +1 to +4 as indicated below the histogram. Data represent the averages of three independent experiments and standard deviations are indicated by error bars.