Increased Drug Affinity as the Mechanistic Basis for Drug Hypersensitivity of a Mutant Type II Topoisomerase

• Published in: The Journal of biological chemistry Vol. 270; no. 47; pp. 28018 - 28021
• Main Authors: Stacie, J. Froelich-Ammon, Burden, D. Andrew, Patchan, Marcia W., Elsea, Sarah H., Thompson, Richard B., Osheroff, Neil
• Format: Journal Article
• Language: English
• Published: United States American Society for Biochemistry and Molecular Biology 24.11.1995
• Subjects: Amino Acid Sequence; Animals; Base Sequence; Binding, Competitive; Chemokines - pharmacology; Cloning, Molecular; DNA Primers; Humans; Interleukin-8 - metabolism; Iodine Radioisotopes; Kinetics; Mice; Molecular Sequence Data; Polymerase Chain Reaction; Receptors, Interleukin - metabolism; Receptors, Interleukin-8B; Recombinant Fusion Proteins - pharmacology; Recombinant Proteins - biosynthesis; Recombinant Proteins - metabolism; Sequence Homology, Amino Acid
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.270.47.28018

Altered sensitivity of topoisomerase II to anticancer drugs profoundly affects the response of eukaryotic cells to these agents. Therefore, several approaches were employed to elucidate the mechanism of drug hypersensitivity of the mutant yeast type II topoisomerase, top2H1012Y. This mutant, which is 5-fold hypersensitive to ellipticine, formed DNA cleavage complexes more rapidly than the wild-type yeast enzyme in the presence of the drug. Conversely, no change in the rate of DNA religation was observed. There was, however, a correlation between increased cleavage rates and enhanced drug binding affinity. The apparent dissociation constant for ellipticine in the mutant topoisomerase II•drug•DNA ternary complex was 5-fold lower than in the wild-type ternary complex. Furthermore, the apparent K value for the mutant binary (topoisomerase II•drug) complex was 2-fold lower than the corresponding wild-type complex, indicating that drug hypersensitivity is intrinsic to the enzyme. These findings strongly suggest that the enhanced ellipticine binding affinity for topoisomerase II is the mechanistic basis for drug hypersensitivity of top2H1012Y.