Strumberg D, Nitiss J L, Rose A, Nicklaus M C, Pommier Y
Laboratory of Molecular Pharmacology, Division of Basic Sciences, NCI, National Institutes of Health, Bethesda, Maryland 20892-4255, USA.
J Biol Chem. 1999 Mar 12;274(11):7292-301. doi: 10.1074/jbc.274.11.7292.
A Ser740 --> Trp mutation in yeast topoisomerase II (top2) and of the equivalent Ser83 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 top2(S740W) in addition to the previously reported C-1 and G+5 for the wild-type top2. The VP-16-induced top2(S740W) cleavage complexes were also more stable. The most stable sites had strong preference for C-1, 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 Ser740 --> 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.
酵母拓扑异构酶II（top2）中的Ser740突变为Trp以及促旋酶中对应的Ser83突变为Trp，会导致对喹诺酮类药物产生抗性，并赋予对依托泊苷（VP-16）的超敏感性。我们对人c-myc基因中top2(S740W)诱导的切割复合物进行了表征。除了对氟喹诺酮CP-115,953具有抗性外，top2(S740W)在存在VP-16、氮杂毒素、安吖啶和米托蒽醌的情况下会诱导产生新的DNA切割位点。对VP-16位点的分析表明，切割模式的变化反映在碱基偏好的改变上。除了先前报道的野生型top2的C-1和G+5外，top2(S740W)在-2位观察到C，在+6位观察到G。VP-16诱导的top2(S740W)切割复合物也更稳定。最稳定的位点对C-1有强烈偏好，而最可逆的位点在-1或-2位没有碱基偏好。在无药物和有钙的情况下也观察到了不同的DNA切割模式。这些结果表明，Ser740突变为Trp改变了top2对DNA的识别，增强了其与DNA的结合，并显著影响了其与抑制剂的相互作用。因此，top2的740位残基对于DNA和药物相互作用似乎都至关重要。
