Cell killing by the F plasmid CcdB protein involves poisoning of DNA-topoisomerase II complexes.
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.
Bernard
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P
Département de Biologie Moléculaire, Université Libre de Bruxelles, Belgium.
Couturier
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M
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
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