Roles of topoisomerases in maintaining steady-state DNA supercoiling in Escherichia coli.
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.
Zechiedrich
E L
EL
Department of Molecular Virology and Microbiology, Baylor College of Medicine, Houston, Texas 77030, USA.
Khodursky
A B
AB
Bachellier
S
S
Schneider
R
R
Chen
D
D
Lilley
D M
DM
Cozzarelli
N R
NR
D027101:DNA Topoisomerase IV; D004264:DNA Topoisomerases, Type I / Q000378:metabolism*; D004250:DNA Topoisomerases, Type II / Q000235:genetics / Q000378:metabolism*; D004269:DNA, Bacterial / Q000378:metabolism*; D004278:DNA, Superhelical / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D008957:Models, Genetic; D009154:Mutation; D009643:Norfloxacin / Q000494:pharmacology; D010957:Plasmids / Q000378:metabolism; D011995:Recombination, Genetic; D059005:Topoisomerase II Inhibitors
D004269:DNA, Bacterial; D004278:DNA, Superhelical; D059005:Topoisomerase II Inhibitors; D027101:DNA Topoisomerase IV; D004264:DNA Topoisomerases, Type I; D004250:DNA Topoisomerases, Type II; D009643:Norfloxacin