ATP-bound topoisomerase II as a target for antitumor drugs

Topoisomerase II (TOP2) poisons interfere with the breakage/reunion reactio n of TOP2 resulting in DNA cleavage. In the current studies, we show that t wo different classes (ATP-sensitive and -insensitive) of TOP2 poisons can b e identified based on their differential sensitivity to the ATP-bound confo rmation of TOP2. First, in the presence of 1 mM ATP or the nonhydrolyzable analog adenosine 5'-(beta,gamma -imino)triphosphate, TOP2-mediated DNA clea vage induced by ATP-sensitive TOP2 poisons (e.g. doxorubicin, etoposide, mi toxantrone, and 4'-(9-acridinylamino)methanesulfon-m-anisidide) was 30-100- fold stimulated, whereas DNA cleavage induced by ATP-insensitive TOP2 poiso ns (e.g. amonafide, batracylin, and menadione) was only slightly (less than 3-fold) affected. In addition, ADP was shown to strongly antagonize TOP2-m ediated DNA cleavage induced by ATP-sensitive but not ATP-insensitive TOP2 poisons. Second, C427A mutant human TOP2 alpha, which exhibits reduced ATPa se activity, was shown to exhibit cross-resistance to all ATP-sensitive but not ATP-insensitive TOP2 poisons. Third, using ciprofloxacin competition a ssay, TOP2-mediated DNA cleavage induced by ATP-sensitive but not ATP-insen sitive poisons was shown to be antagonized by ciprofloxacin. These results suggest that ATP-bound TOP2 may be the specific target of ATP-sensitive TOP 2 poisons. Using Lac repressor-operator complexes as roadblocks, we show th at ATP-bound TOP2 acts as a circular clamp capable of entering DNA ends and sliding on unobstructed duplex DNA.