The mechanism of inhibition of topoisomerase IV by quinolone antibacterials.
Topoisomerase IV (Topo IV) is a mediator of quinolone toxicity in bacteria. In this work, we demonstrate that norfloxacin, a model quinolone, converts Escherichia coli Topo IV into a poisonous adduct on DNA as opposed to inhibiting topoisomerase activity. Norfloxacin inhibition of Topo IV induces a slow decline in DNA synthesis that parallels cell death. Treatment of cells with a lethal concentration of the antibacterial did not block chromosome segregation, the phenotype of catalytic inhibition of Topo IV. Instead, norfloxacin causes DNA damage, as evidenced by the induction of the SOS pathway for DNA repair; the increase in susceptibility to the drug by mutations in genes for DNA repair pathways including recA, recB, and uvrD; and the efficient detergent-induced linearization of plasmid DNA in drug-treated cells. Wild-type and drug-resistant alleles of Topo IV are co-dominant, but we find that mutations in recA, seqA, or gyrB result in unconditional dominance of the sensitive allele, the characteristic of a poisoning mode of inhibition. These mutations either compromise chromosome integrity or force Topo IV to play a more active role in DNA unlinking in front of the replication fork. We interpret our results in terms of distinct but complementary roles of Topo IV and gyrase in DNA replication.
Department of Molecular and Cell Biology, University of California, Berkeley, California 94720-3204, USA.
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Khodursky
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Cozzarelli
D000251:Adenosine Triphosphatases / Q000235:genetics; D000890:Anti-Infective Agents / Q000494:pharmacology*; D002876:Chromosomes, Bacterial / Q000187:drug effects; D018736:DNA Adducts; D004249:DNA Damage; D004265:DNA Helicases; D004260:DNA Repair / Q000187:drug effects / Q000235:genetics; D004261:DNA Replication / Q000187:drug effects; D027101:DNA Topoisomerase IV; D004250:DNA Topoisomerases, Type II / Q000187:drug effects / Q000378:metabolism; D004926:Escherichia coli / Q000187:drug effects* / Q000201:enzymology; D029968:Escherichia coli Proteins; D043211:Exodeoxyribonuclease V; D005090:Exodeoxyribonucleases / Q000235:genetics; D008957:Models, Genetic; D009154:Mutation; D009643:Norfloxacin / Q000494:pharmacology*; D010957:Plasmids / Q000096:biosynthesis; D011485:Protein Binding; D011938:Rec A Recombinases / Q000235:genetics; D013014:SOS Response, Genetics; D059005:Topoisomerase II Inhibitors
D000890:Anti-Infective Agents; D018736:DNA Adducts; D029968:Escherichia coli Proteins; D059005:Topoisomerase II Inhibitors; D011938:Rec A Recombinases; D005090:Exodeoxyribonucleases; D043211:Exodeoxyribonuclease V; C468322:exodeoxyribonuclease V, E coli; D000251:Adenosine Triphosphatases; C506746:UvrD protein, E coli; D004265:DNA Helicases; D027101:DNA Topoisomerase IV; D004250:DNA Topoisomerases, Type II; D009643:Norfloxacin