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Yeast Topoisomerase II Mutants Resistant to Anti-Topoisomerase Agents: Identification and Characterization of New Yeast Topoisomerase II Mutants Selected for Resistance to Etoposide¹

Developmental Therapeutics Section, Division of Hematology/Oncology, Childrens Hospital of Los Angeles [Y-X. L., M. J., J. L. N.], and Department of Biochemistry and Molecular Biology, University of Southern California Medical School [Y. H., Y. Y., J. L. N.], Los Angeles, California 90027

We describe a system that allows us to easily isolate and characterize mutants in yeast topoisomerase II that are resistant to antitumor agents that target this enzyme. The system uses yeast strains that are sensitive to those agents and that carry temperature-sensitive top2 mutations. The temperature-sensitive mutation allows the isolation of recessive drug-resistant mutations. The mutagenized TOP2 gene we have used is under the control of the yeast DED1 promoter; this overexpression of TOP2 is designed to avoid isolating mutants that are drug resistant solely because the mutated topoisomerase II has low enzymatic activity. We describe three mutants that we isolated using this system. Two of the three mutants show resistance to etoposide and amsacrine, while the third mutant is partially resistant to etoposide and fluoroquinolones but not to amsacrine. DNA sequence changes have been identified in all of these mutant TOP2 genes. The mutant with partial resistance to etoposide and fluoroquinolones has an amino acid change at position 738 of TOP2, which is three amino acids from the site homologous to Ser₈₃ of E. coli gyrA, an amino acid which had previously been shown to be an important target for resistance to quinolones in bacteria. One of the alleles that confers resistance to both etoposide and amsacrine, top2-103, has changes in amino acid 824 and amino acid 1186 of TOP2. Reconstruction of the mutations by oligonucleotide-directed mutagenesis demonstrates that the change at amino acid 824 is responsible for the drug resistance of this allele.

¹ This work was supported by Grant CA 52814 from the National Cancer Institute and by the Neil Bogart Laboratory of the Martell Foundation for Leukemia, Cancer, and AIDS. J. L. N. was a Special Fellow of the Leukemia Society of America during the course of this work.

² To whom requests for reprints should be addressed, at Division of Hematology/Oncology, MS 126, Childrens Hospital of Los Angeles, 4650 Sunset Blvd., Los Angeles, CA 90027.

Received 12/17/93. Accepted 3/29/94.

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