PT  - JOURNAL ARTICLE
AU  - Sandhiya Patel
AU  - Elen Jazrawi
AU  - Andrew M. Creighton
AU  - Caroline A. Austin
AU  - L. Mark Fisher
TI  - Probing the Interaction of the Cytotoxic Bisdioxopiperazine ICRF-193 with the Closed Enzyme Clamp of Human Topoisomerase IIα
AID  - 10.1124/mol.58.3.560
DP  - 2000 Sep 01
TA  - Molecular Pharmacology
PG  - 560--568
VI  - 58
IP  - 3
4099  - http://molpharm.aspetjournals.org/content/58/3/560.short
4100  - http://molpharm.aspetjournals.org/content/58/3/560.full
SO  - Mol Pharmacol2000 Sep 01; 58
AB  - 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 IIα alleles resistant to ICRF-193. Mutations in topoisomerase IIα 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 IIα.