Wang H, Mao Y, Zhou N, Hu T, Hsieh T S, Liu L F
Department of Pharmacology, University of Medicine and Dentistry of New Jersey/Robert Wood Johnson Medical School, Piscataway, New Jersey 08854, USA.
J Biol Chem. 2001 May 11;276(19):15990-5. doi: 10.1074/jbc.M011143200. Epub 2001 Feb 23.
Topoisomerase II (TOP2) poisons interfere with the breakage/reunion reaction of TOP2 resulting in DNA cleavage. In the current studies, we show that two different classes (ATP-sensitive and -insensitive) of TOP2 poisons can be identified based on their differential sensitivity to the ATP-bound conformation of TOP2. First, in the presence of 1 mm ATP or the nonhydrolyzable analog adenosine 5'-(beta,gamma-imino)triphosphate, TOP2-mediated DNA cleavage induced by ATP-sensitive TOP2 poisons (e.g. doxorubicin, etoposide, mitoxantrone, and 4'-(9-acridinylamino)methanesulfon-m-anisidide) was 30-100-fold stimulated, whereas DNA cleavage induced by ATP-insensitive TOP2 poisons (e.g. amonafide, batracylin, and menadione) was only slightly (less than 3-fold) affected. In addition, ADP was shown to strongly antagonize TOP2-mediated DNA cleavage induced by ATP-sensitive but not ATP-insensitive TOP2 poisons. Second, C427A mutant human TOP2alpha, which exhibits reduced ATPase activity, was shown to exhibit cross-resistance to all ATP-sensitive but not ATP-insensitive TOP2 poisons. Third, using ciprofloxacin competition assay, TOP2-mediated DNA cleavage induced by ATP-sensitive but not ATP-insensitive poisons was shown to be antagonized by ciprofloxacin. These results suggest that ATP-bound TOP2 may be the specific target of ATP-sensitive TOP2 poisons. Using Lac repressor-operator complexes as roadblocks, we show that ATP-bound TOP2 acts as a circular clamp capable of entering DNA ends and sliding on unobstructed duplex DNA.
拓扑异构酶II（TOP2）毒剂干扰TOP2的断裂/重连反应，导致DNA裂解。在当前研究中，我们表明，基于对TOP2的ATP结合构象的不同敏感性，可以鉴定出两类不同的（对ATP敏感和不敏感的）TOP2毒剂。首先，在存在1 mM ATP或不可水解类似物腺苷5'-（β，γ-亚氨基）三磷酸的情况下，由ATP敏感的TOP2毒剂（如阿霉素、依托泊苷、米托蒽醌和4'-（9-吖啶基氨基）甲磺酰基间茴香胺）诱导的TOP2介导的DNA裂解受到30至100倍的刺激，而由ATP不敏感的TOP2毒剂（如氨萘非特、巴曲霉素和甲萘醌）诱导的DNA裂解仅受到轻微影响（小于3倍）。此外，ADP被证明强烈拮抗由ATP敏感但非ATP不敏感的TOP2毒剂诱导的TOP2介导的DNA裂解。其次，表现出ATP酶活性降低的C427A突变型人TOP2α对所有ATP敏感但非ATP不敏感的TOP2毒剂表现出交叉抗性。第三，使用环丙沙星竞争试验，由ATP敏感但非ATP不敏感的毒剂诱导的TOP2介导的DNA裂解被环丙沙星拮抗。这些结果表明，ATP结合的TOP2可能是ATP敏感的TOP2毒剂的特异性靶点。使用乳糖阻遏物-操纵子复合物作为障碍物，我们表明ATP结合的TOP2作为一种环状夹，能够进入DNA末端并在无阻碍的双链DNA上滑动。
