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 (sigma) 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 sigma 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 sigma = -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 (sigma = -0.09), greatly stimulating transcription from the supercoiling sensitive leu-500 promoter and increasing the number of supercoils trapped by lambda integrase site-specific recombination.
Highlights
DNA supercoiling is essential for viability and is tightly regulated in the cell
We focus on the properties of the topoisomerases of Escherichia coli
We show that topoisomerase I and topoisomerase IV counter gyrase to maintain DNA supercoiling and that each topoisomerase has distinct roles
Summary
⌬(topA-cysB)204 acrA13 gyrB225 Hfr thi F-metB argE ilv tna gyrB134 cour tsb C600 except zei-723::Tn10 parCL80kan. RS2 except kan RS2 except parCL80kan RS2 except parCK84kan KL16 except zei-3143::Tn10kan. The decatenation of replication or recombination intermediates by topoisomerase IV is facilitated by the (Ϫ) supercoiling activity of gyrase [15, 16]. The wild-type, steady-state supercoiling level, , of around Ϫ0.06 to Ϫ0.075 had long been thought to be regulated only by the opposing activities of topoisomerase I relaxing and gyrase introducing (Ϫ) supercoils. (iv) In E. coli, when gyrase activity is blocked, integrase (Int) recombination is reduced approximately 7-fold when topoisomerase IV remains functional compared with when topoisomerase IV is blocked, showing that topoisomerase IV is able to relax DNA [16]. We show that topoisomerase I and topoisomerase IV counter gyrase to maintain DNA supercoiling and that each topoisomerase has distinct roles
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