Probing the interaction of the cytotoxic bisdioxopiperazine ICRF-193 with the closed enzyme clamp of human topoisomerase II alpha

Topoisomerase II is an ATP-operated protein clamp that captures a DNA helix and transports it through another DNA duplex, allowing chromosome segregat ion at mitosis. A number of cytotoxic bisdioxopiperazines such as ICRF-193 target topoisomerase II by binding and trapping the closed enzyme clamp. To investigate this unusual mode of action, we have used yeast to select plas mid-borne human topoisomerase II alpha alleles resistant to ICRF-193. Mutat ions in topoisomerase II alpha of Leu-169 to Phe (L169F) (in the N-terminal ATPase domain) and Ala-648 to Pro (A648P) (in the core domain) were identi fied as conferring >50-fold and 5-fold resistance to ICRF-193 in vivo, resp ectively. The L169F mutation, located next to the Walker A box ATP-binding sequence, resulted in a mutant enzyme displaying ICRF-193-resistant topoiso merase and ATPase activities and whose closed clamp was refractory to ICRF- 193-mediated trapping as an annulus on closed circular DNA. These data impl y that the mutation interferes directly with ICRF-193 binding to the N-term inal ATPase gate. In contrast, the A648P enzyme displayed topoisomerase act ivities exhibiting wild-type sensitivity to ICRF-193. We suggest that the i nefficient trapping of the A648P closed clamp results either from the obser ved increased ATP requirement, or more likely, from lowered salt stability, perhaps involving destabilization of ICRF-193 interactions with the B'-B' interface in the core domain. These results provide evidence for at least t wo different phenotypic classes of ICRF-193 resistance mutations and sugges t that bisdioxopiperazine action involves the interplay of both the ATPase and core domains of topoisomerase II alpha.