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 equ ivalent Ser(83) in gyrase results in resistance to quinolones and confers h ypersensitivity to etoposide (VP-16), We characterized the cleavage complex es induced by the top2(S740W) in the human c-myc gene. In addition to resis tance to the fluoroquinolone CP-115,953, top2(S740W) induced novel DNA clea vage sites in the presence of VP-16, azatoxin, amsacrine, and mitoxantrone, Analysis of the VP-16 sites indicated that the changes in the cleavage pat tern 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 prev iously reported C-1 and G+5 for the wildtype top2, The VP-16-induced top(2S 740W) cleavage complexes were also more stable. The most stable sites had s trong preference for C-l, whereas the most reversible sites showed no base preference at positions -1 or -2, Different patterns of DNA cleavage were a lso observed in the absence of drug and in the presence of calcium. These r esults indicate that the Ser(740) --> Trp mutation alters the DNA recogniti on of top2, enhances its DNA binding, and markedly affects its interactions with inhibitors. Thus, residue 740 of top2 appears critical for both DNA a nd drug interactions.