We have demonstrated that, in Escherichia coli, quinolone antimicrobial agents target topoisomerase IV (topo IV). The inhibition of topo IV becomes apparent only when gyrase is mutated to quinolone resistance. In such mutants, these antibiotics caused accumulation of replication catenanes, which is diagnostic of a loss of topo IV activity. Mutant forms of topo IV provided an additional 10-fold resistance to quinolones and prevented drug-induced catenane accumulation. Drug inhibition of topo IV differs from that of gyrase. (i) Wild-type topo IV is not dominant over the resistant allele. (ii) Inhibition of topo IV leads to only a slow stop in replication. (iii) Inhibition of topo IV is primarily bacteriostatic. These differences may result from topo IV acting behind the replication fork, allowing for repair of drug-induced lesions. We suggest that this and a slightly higher intrinsic resistance of topo IV make it secondary to gyrase as a quinolone target. Our results imply that the quinolone binding pockets of gyrase and topo IV are similar and that substantial levels of drug resistance require mutations in both enzymes.The quinolone antibacterial agents have had a long and important history in the clinic and in basic research. The biological activity of the founding member of the group, nalidixic acid, was discovered in 1965 (1). Successive generations of drugs have brought orders of magnitude increases in efficacy and they are now one of the most widely used classes of antibacterial agents. The primary target of these drugs in Escherichia coli was established in 1977 as DNA gyrase, a type-2 topoisomerase (2, 3). The critical role of gyrase is to unlink chromosomal DNA during its replication by the introduction of negative supercoils (4, 5). The quinolones inhibit gyrase activity in vitro and a single amino acid change can cause a 10-to 100-fold decrease in drug sensitivity (6).The potency of the quinolones is caused by their striking mode of action. It was shown in 1979 that they block DNA synthesis not by depriving the cell of gyrase but by converting gyrase to a poison of replication (7). Anticancer drugs that inhibit human type-2 topoisomerases also convert their targets into poisons (8). The poisoning is mediated by trapping of an intermediate in topoisomerizatiorn, which ultimately leads to a double-strand break of DNA (9).A number of results have implicated secondary targets for quinolones. Chief among these is the multistep resistance to quinolones that occurs clinically, in which mutations in gyrase are only an initial step (10). A second bacterial type-2 topoisomerase was discovered with a sequence similar to that of gyrase, particularly in the region responsible for drug sensitivity (11). This essential enzyme, topoisomerase IV (topo IV), is required for the terminal stages of unlinking of DNA during replication (11,12). The first evidence that topo IV might be a quinolone target was the demonstration that it is inhibited by quinolones in vitro almost as well as gyrase (13). Recently,The public...

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 () 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 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 ‫؍‬ ؊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 ( ‫؍‬ ؊0.09), greatly stimulating transcription from the supercoiling sensitive leu-500 promoter and increasing the number of supercoils trapped by integrase site-specific recombination.

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For a cell to complete DNA replication, every link between the Watson-Crick strands must be removed by topoisomerases. Previously, we reported that the inhibition of topoisomerase IV (topo IV) leads to the accumulation of catenated plasmid replicons to a steady-state level of -10%. Using pulse labeling with [3~]thymidine in Escherichia coli, we have found that in the absence of top0 IV activity, nearly all newly synthesized plasmid DNA is catenated. Pulse-chase protocols revealed that catenanes are metabolized even in the absence of top0 IV and that the residual turnover is carried out by DNA gyrase at a rate of -O.Ol/sec. Using extremely short pulse-labeling times, we identified significant amounts of replication catenanes in wild-type cells. The rate of catenane unlinking in wild-type cells by the combined activities of top0 IV and DNA gyrase was -l/sec. Therefore, gyrase is 100-fold less efficient than top0 IV in plasmid replicon decatenation in vivo. This may explain why a fully functional gyrase cannot prevent the catenation of newly synthesized plasmid DNA and the partition phenotype of top0 IV mutants. We conclude that catenanes are kinetic intermediates in DNA replication and that the essential role of top0 IV is to unlink daughter replicons.

By regulating access to the genetic code, DNA supercoiling strongly affects DNA metabolism. Despite its importance, however, much about supercoiled DNA (positively supercoiled DNA, in particular) remains unknown. Here we use electron cryo-tomography together with biochemical analyses to investigate structures of individual purified DNA minicircle topoisomers with defined degrees of supercoiling. Our results reveal that each topoisomer, negative or positive, adopts a unique and surprisingly wide distribution of three-dimensional conformations. Moreover, we uncover striking differences in how the topoisomers handle torsional stress. As negative supercoiling increases, bases are increasingly exposed. Beyond a sharp supercoiling threshold, we also detect exposed bases in positively supercoiled DNA. Molecular dynamics simulations independently confirm the conformational heterogeneity and provide atomistic insight into the flexibility of supercoiled DNA. Our integrated approach reveals the three-dimensional structures of DNA that are essential for its function.