Human cells express two isoforms of topoisomerase II, alpha and beta, that are both targeted by anticancer drugs. To investigate acridine resistance mediated by topoisomerase IIbeta, we used a forced molecular evolution approach. A library of mutated topoisomerase IIbeta cDNAs was generated by hydroxylamine mutagenesis and was transformed into the yeast JN394 top2-4. Methyl N-(4'-(9-acridinylamino)-phenyl)carbamate hydrochloride (AMCA) selection identified a resistant transformant able to grow in media containing 76 microg/ml AMCA. Topoisomerase IIbeta with a glutamic acid-to-lysine substitution at position 522 was responsible for the approximately 10-fold resistance to AMCA. The transformant was cross-resistant to methyl N-(4'-(9-acridinylamino)-3-methoxy-phenyl) methane sulfonamide (mAMSA) and mAMCA but hypersensitive to etoposide and ellipticine. In vitro, the betaE522K protein was unable to support acridine-stimulated DNA cleavage, suggesting that resistance to these acridines is caused by reduced drug-stimulated DNA cleavage. However, betaE522K showed DNA cleavage with etoposide, and the cleavable complexes formed with etoposide showed greater stability, thus accounting for the hypersensitivity to etoposide. Drug-independent cleavage of an oligonucleotide by betaE522K was reduced compared with the wild-type enzyme. Decatenation and relaxation activities were reduced to 52 and 61% of the wild-type levels, which may explain the slower growth of yeast strain JN394top2-4 expressing betaE522K at the nonpermissive temperature. This study confirms that topoisomerase IIbeta is a target for AMCA and that resistance to AMCA can be mediated by a point mutation at Glu522 in topoisomerase IIbeta. Residue 522 lies within a Rossmann fold in the B' subfragment of topoisomerase II, a region previously implicated in drug interactions.