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 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.
Crisona
N J
NJ
Department of Molecular and Cell Biology, University of California, Berkeley, California 94720, USA.
Strick
T R
TR
Bensimon
D
D
Croquette
V
V
Cozzarelli
N R
NR
D027101:DNA Topoisomerase IV; D004250:DNA Topoisomerases, Type II / Q000378:metabolism*; D004278:DNA, Superhelical / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology*; D007536:Isomerism; D008957:Models, Genetic; D009690:Nucleic Acid Conformation; D011485:Protein Binding; D013379:Substrate Specificity
D004278:DNA, Superhelical; D027101:DNA Topoisomerase IV; D004250:DNA Topoisomerases, Type II