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

Abstract

We show that positively supercoiled [(+) SC] DNA is the preferred substrate for Escherichia coli topoisomerase IV (topo IV). We measured topo IV relaxation of (−) and (+) supercoils in real time on single, tethered DNA molecules 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 preferential cleavage of (+) SC DNA in a competition experiment showed that substrate discrimination can take place prior to strand passage in the presence or absence of ATP. We propose that topo IV discriminates between (−) and (+) supercoiled DNA by recognition of the geometry of (+) SC DNA. Our results explain how topo IV can rapidly remove (+) supercoils to support DNA replication without relaxing the essential (−) supercoils of the chromosome. They also 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.

Keywords

• Topoisomerase IV
• positively supercoiled DNA
• single-molecule biophysics
• type II topoisomerases
• DNA replication