Roles of topoisomerases in maintaining steady-state DNA supercoiling in Escherichia coli

E. Lynn Zechiedrich, Arkady B. Khodursky, Sophie Bachellier, Robert Schneider, Dongrong Chen, David M J Lilley, Nicholas R. Cozzarelli

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Abstract

DNA supercoiling is essential for bacterial cell survival. We demonstrated that DNA topoisomerase IV, acting in concert with topoisomerase I and gyrase, makes an important contribution to the steady-state level of supercoiling in Escherichia coli. Following inhibition of gyrase, topoisomerase IV alone relaxed plasmid DNA to a final supercoiling density (σ) of -0.015 at an initial rate of 0.8 links min-1. Topoisomerase I relaxed DNA at a faster rate, 5 links min-1, but only to a σ of -0.05. Inhibition of topoisomerase IV in wild-type cells increased supercoiling to approximately the same level as in a mutant lacking topoisomerase I activity (to σ = -0.08). The role of topoisomerase IV was revealed by two functional assays. Removal of both topoisomerase I and topoisomerase IV caused the DNA to become hyper-negatively supercoiled (σ = -0.09), greatly stimulating transcription from the supercoiling sensitive leu-500 promoter and increasing the number of supercoils trapped by λ integrase site-specific recombination.

Original languageEnglish (US)
Pages (from-to)8103-8113
Number of pages11
JournalJournal of Biological Chemistry
Volume275
Issue number11
DOIs
StatePublished - Mar 17 2000

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    Zechiedrich, E. L., Khodursky, A. B., Bachellier, S., Schneider, R., Chen, D., Lilley, D. M. J., & Cozzarelli, N. R. (2000). Roles of topoisomerases in maintaining steady-state DNA supercoiling in Escherichia coli. Journal of Biological Chemistry, 275(11), 8103-8113. https://doi.org/10.1074/jbc.275.11.8103