Mutation of a conserved serine residue in a quinolone-resistant type II topoisomerase alters the enzyme-DNA and drug interactions

A Ser(740) --> Trp mutation in yeast topoisomerase II (top2) and of the equivalent Ser(83) in gyrase results in resistance to quinolones and confers hypersensitivity to etoposide (VP-16), We characterized the cleavage complexes induced by the top2(S740W) in the human c-myc gene. In addition to resistance to the fluoroquinolone CP-115,953, top2(S740W) induced novel DNA cleavage sites in the presence of VP-16, azatoxin, amsacrine, and mitoxantrone, Analysis of the VP-16 sites indicated that the changes in the cleavage pattern were reflected by alterations in base preference. C at position -2 and G at position +6 were observed for the top(2S740W) in addition to the previously reported C-1 and G+5 for the wildtype top2, The VP-16-induced top(2S740W) cleavage complexes were also more stable. The most stable sites had strong preference for C-l, whereas the most reversible sites showed no base preference at positions -1 or -2, Different patterns of DNA cleavage were also observed in the absence of drug and in the presence of calcium. These results indicate that the Ser(740) --> Trp mutation alters the DNA recognition of top2, enhances its DNA binding, and markedly affects its interactions with inhibitors. Thus, residue 740 of top2 appears critical for both DNA and drug interactions.