Preferential relaxation of positively supercoiled DNA by E-coli topoisomerase IV in single-molecule and ensemble measurements

We show that positively supercoiled [(+) SC] DNA is the preferred substrate for Escherichia coli topoisomerase IV (topo IV). We measured topo IV relax ation of (-) and (+) supercoils in real time on single, tethered DNA molecu les to complement ensemble experiments. We find that the preference for (+) SC DNA is complete at low enzyme concentration. Otherwise, topo IV relaxed (+) supercoils at a 20-fold faster rate than (-) supercoils, due primarily to about a 10-fold increase in processivity with (+) SC DNA. The preferent ial cleavage of (+) SC DNA in a competition experiment showed that substrat e discrimination can take place prior to strand passage in the presence or absence of ATP. We propose that topo IV discriminates between (-) and (+) s upercoiled DNA by recognition of the geometry of (+) SC DNA. Our results ex plain how topo IV can rapidly remove (+) supercoils to support DNA replicat ion without relaxing the essential (-) supercoils of the chromosome. They a lso show that the rate of supercoil relaxation by topo IV is several orders of magnitude faster than hitherto appreciated, so that a single enzyme may suffice at each replication fork.