Vol. 58, Issue 3, 560-568, September 2000

Probing the Interaction of the Cytotoxic Bisdioxopiperazine ICRF-193 with the Closed Enzyme Clamp of Human Topoisomerase II

Sandhiya Patel, Elen Jazrawi, Andrew M. Creighton, Caroline A. Austin, and L. Mark Fisher

Molecular Genetics Group, Department of Biochemistry and Immunology, St. George's Hospital Medical School, University of London, London, United Kingdom (S.P., E.J., L.M.F.); Medicinal Chemistry Laboratory, Department of Reproductive Physiology, St. Bartholomew's Hospital Medical College, London, United Kingdom (A.M.C.); and Department of Biochemistry and Genetics, University of Newcastle, Newcastle-upon-Tyne, United Kingdom (C.A.A.)

Topoisomerase II is an ATP-operated protein clamp that captures a DNA helix and transports it through another DNA duplex, allowing chromosome segregation 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 plasmid-borne human topoisomerase II alleles resistant to ICRF-193. Mutations in topoisomerase II of Leu-169 to Phe (L169F) (in the N-terminal ATPase domain) and Ala-648 to Pro (A648P) (in the core domain) were identified as conferring >50-fold and 5-fold resistance to ICRF-193 in vivo, respectively. The L169F mutation, located next to the Walker A box ATP-binding sequence, resulted in a mutant enzyme displaying ICRF-193-resistant topoisomerase and ATPase activities and whose closed clamp was refractory to ICRF-193-mediated trapping as an annulus on closed circular DNA. These data imply that the mutation interferes directly with ICRF-193 binding to the N-terminal ATPase gate. In contrast, the A648P enzyme displayed topoisomerase activities exhibiting wild-type sensitivity to ICRF-193. We suggest that the inefficient trapping of the A648P closed clamp results either from the observed 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 two different phenotypic classes of ICRF-193 resistance mutations and suggest that bisdioxopiperazine action involves the interplay of both the ATPase and core domains of topoisomerase II.