Increased sensitivity to quinolone antibacterials can be engineered in human topoisomerase II alpha by selective mutagenesis

A potential region of drug-DNA interaction in the A subunit of DNA gyrase h as previously been identified from crystallographic studies. The local amin o acid sequence has been compared with similar regions in yeast topoisomera se II and human topoisomerase II alpha. Three nonconserved, potentially sol vent-accessible residues at positions 762, 763 and 766 in human topoisomera se II alpha lie between well-conserved regions. The corresponding residues in GyrA (83, 84 and 87) have a high frequency of mutation in quinolone-resi stant bacteria. Mutations in human topoisomerase II alpha have been generat ed in an attempt to engineer ciprofloxacin sensitivity into this enzyme: M7 62S, S763A and M766D (each mutated to the identical amino acid present in g yrase), along with an M762S/S763A double mutant and a triple mutant. These enzymes were introduced into a temperature-sensitive yeast strain, deficien t in topoisomerase II, for in vivo studies, and were overproduced for in vi tro 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 DN A and had reduced catalytic activity. The triple mutation confers a three-f old increase in sensitivity to ciprofloxacin in vitro and similar sensitivi ties to a range of other quinolones. The activity of the quinolone CP-115,9 53, a bacterial and eukaryotic topoisomerase II poison, was unaffected by a ny of these mutations. Mutations in this region were found to increase the sensitivity of the enzyme to the DNA intercalating anti-tumour agents m-AMS A and ellipticine, but confer resistance to the non-intercalating agents et oposide, teniposide and merbarone, an effect that was maximal in the triple mutant. We have therefore shown the importance of this region in determini ng the sensitivity of topoisomerase II to drugs and have engineered increas ed sensitivity to quinolones. (C) 2000 Academic Press.