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

Publication Type:

Journal Article

Source:

GENES & DEVELOPMENT, COLD SPRING HARBOR LAB PRESS, Volume 14, Number 22, 1 BUNGTOWN RD, PLAINVIEW, NY 11724 USA, p.2881-2892 (2000)

Keywords:

CLEAVAGE; ENZYME; ESCHERICHIA-COLI; GYRASE; MECHANISM; PURIFICATION; REPLICATION; TOPOLOGY; TRANSPORT; UNLINKING

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.