title: Cell killing by the F plasmid CcdB protein involves poisoning of DNA-topoisomerase II complexes.
abstract: In Escherichia coli, the miniF plasmid CcdB protein is responsible for cell death when its action is not prevented by polypeptide CcdA. We report the isolation, localization, sequencing and properties of a bacterial mutant resistant to the cytotoxic activity of the CcdB protein. This mutation is located in the gene encoding the A subunit of topoisomerase II and produces an Arg462----Cys substitution in the amino acid sequence of the GyrA polypeptide. Hence, the mutation was called gyrA462. We show that in the wild-type strain, the CcdB protein promotes plasmid linearization; in the gyrA462 strain, this double-stranded DNA cleavage is suppressed. This indicates that the CcdB protein is responsible for gyrase-mediated double-stranded DNA breakage. CcdB, in the absence of CcdA, induces the SOS pathway. SOS induction is a biological response to DNA-damaging agents. We show that the gyrA462 mutation suppresses this SOS activation, indicating that SOS induction is a consequence of DNA damages promoted by the CcdB protein on gyrase-DNA complexes. In addition, we observe that the CcdBS sensitive phenotype dominates over the resistant phenotype. This is better explained by the conversion, in gyrA+/gyrA462 merodiploid strains, of the wild-type gyrase into a DNA-damaging agent. These results strongly suggest that the CcdB protein, like quinolone antibiotics and a variety of antitumoral drugs, is a DNA topoisomerase II poison. This is the first proteinic poison-antipoison mechanism that has been found to act via the DNA topoisomerase II.
authors.affiliation: Département de Biologie Moléculaire, Université Libre de Bruxelles, Belgium.
authors.forename: P
authors.initials: P
authors.lastname: Bernard
authors.forename: M
authors.initials: M
authors.lastname: Couturier
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000378:metabolism*; D001427:Bacterial Toxins / Q000378:metabolism*; D016923:Cell Death; D003603:Cytotoxins / Q000378:metabolism; D004249:DNA Damage; D027081:DNA Gyrase; D004250:DNA Topoisomerases, Type II / Q000235:genetics / Q000378:metabolism*; D004269:DNA, Bacterial / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000166:cytology / Q000235:genetics / Q000378:metabolism*; D005144:F Factor; D005798:Genes, Bacterial; D046911:Macromolecular Substances; D009154:Mutation; D010957:Plasmids; D011485:Protein Binding; D011995:Recombination, Genetic; D015183:Restriction Mapping; D013014:SOS Response, Genetics
chemical_list: D001426:Bacterial Proteins; D001427:Bacterial Toxins; C068356:CcdB protein, Plasmid F; D003603:Cytotoxins; D004269:DNA, Bacterial; D046911:Macromolecular Substances; D027081:DNA Gyrase; D004250:DNA Topoisomerases, Type II