Figure 5: Mapping Bal-31 cleavage.
To determine whether Bal-31 cleavage occurs at multiple sites or at a preferred site, the ΔLk=−6 topoisomer was cleaved with Bal-31 and various restriction enzymes. (a) Products were separated by agarose gel electrophoresis. Left, (lanes 1–5), control reactions, mc336 (approximately equal mixture of ΔLk=−2 and ΔLk=−3 topoisomers) with combinations of the various restriction enzymes (as indicated) to generate fragments of known DNA lengths. Right, (lanes 6–9), ΔLk=−6 topoisomer cleaved first with Bal-31, followed by a restriction enzyme (as indicated). Mr1: 100 bp DNA ladder, Mr2: Low molecular weight DNA ladder. (b) Map of the minicircle sequence showing the positions of the restriction enzymes used, the estimated location of Bal-31 cleavage (with parentheses indicating the range), and the location of the observed base-pair breaking in MD simulation of the ΔLk=−3 topoisomer.
