Preferential relaxation of positively supercoiled DNA by E. coli topoisomerase IV in single-molecule and ensemble measurements (original) (raw)
- Nancy J. Crisona1,
- Terence R. Strick2,
- David Bensimon2,
- Vincent Croquette2, and
- Nicholas R. Cozzarelli1,3
- 1Department of Molecular and Cell Biology, University of California, Berkeley, California 94720, USA; 2Laboratoire de Physique Statistique de l'ENS, associé aux universités Paris VI et VII, 75231 Paris Cedex 05, France
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.
- Topoisomerase IV
- positively supercoiled DNA
- single-molecule biophysics
- type II topoisomerases
- DNA replication
Footnotes
↵3 Corresponding author.
E-MAIL ncozzare{at}socrates.berkeley.edu; FAX (510) 643-1079.
Article and publication are atwww.genesdev.org/cgi/doif/10.1101/gad.838900
- Received July 27, 2000.
- Accepted October 4, 2000.
Cold Spring Harbor Laboratory Press