Increased sensitivity to quinolone antibacterials can be engineered in human topoisomerase IIalpha by selective mutagenesis.
A potential region of drug-DNA interaction in the A subunit of DNA gyrase has previously been identified from crystallographic studies. The local amino acid sequence has been compared with similar regions in yeast topoisomerase II and human topoisomerase IIalpha. Three non- conserved, potentially solvent-accessible residues at positions 762, 763 and 766 in human topoisomerase IIalpha lie between well-conserved regions. The corresponding residues in GyrA (83, 84 and 87) have a high frequency of mutation in quinolone-resistant bacteria. Mutations in human topoisomerase IIalpha have been generated in an attempt to engineer ciprofloxacin sensitivity into this enzyme: M762S, S763A and M766D (each mutated to the identical amino acid present in gyrase), along with an M762S/S763A double mutant and a triple mutant. These enzymes were introduced into a temperature-sensitive yeast strain, deficient in topoisomerase II, for in vivo studies, and were overproduced for in vitro studies. The M766D mutation renders the enzyme incapable of supporting the temperature-sensitive strain at a non-permissive temperature. However, both M766D and the triple mutant enzymes can be overproduced and are fully active in vitro. The double mutant was impaired in its ability to cleave DNA and had reduced catalytic activity. The triple mutation confers a three-fold increase in sensitivity to ciprofloxacin in vitro and similar sensitivities to a range of other quinolones. The activity of the quinolone CP-115,953, a bacterial and eukaryotic topoisomerase II poison, was unaffected by any of these mutations. Mutations in this region were found to increase the sensitivity of the enzyme to the DNA intercalating anti-tumour agents m-AMSA and ellipticine, but confer resistance to the non-intercalating agents etoposide, teniposide and merbarone, an effect that was maximal in the triple mutant. We have therefore shown the importance of this region in determining the sensitivity of topoisomerase II to drugs and have engineered increased sensitivity to quinolones.
Hammonds
T R
TR
Department of Biochemistry, University of Leicester, Leicester, LE1 7RH, UK. ony@le.ac.uk
Foster
S R
SR
Maxwell
A
A
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution / Q000235:genetics; D000890:Anti-Infective Agents / Q000494:pharmacology*; D000970:Antineoplastic Agents / Q000494:pharmacology; D002384:Catalysis / Q000187:drug effects; D002939:Ciprofloxacin / Q000494:pharmacology; D004250:DNA Topoisomerases, Type II / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D004278:DNA, Superhelical / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D005816:Genetic Complementation Test; D006801:Humans; D007364:Intercalating Agents / Q000494:pharmacology; D008826:Microbial Sensitivity Tests; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics*; D009690:Nucleic Acid Conformation / Q000187:drug effects; D010957:Plasmids / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011487:Protein Conformation; D015202:Protein Engineering; D012441:Saccharomyces cerevisiae / Q000187:drug effects / Q000201:enzymology / Q000235:genetics; D013696:Temperature; D059005:Topoisomerase II Inhibitors
D000890:Anti-Infective Agents; D000970:Antineoplastic Agents; D004278:DNA, Superhelical; D007364:Intercalating Agents; D059005:Topoisomerase II Inhibitors; D002939:Ciprofloxacin; D004250:DNA Topoisomerases, Type II