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J Biol Chem, Vol. 273, Issue 44, 29086-29092, October 30, 1998

A Mutant Yeast Topoisomerase II (top2G437S) with Differential Sensitivity to Anticancer Drugs in the Presence and Absence of ATP

Michelle Sabourin, Jo Ann Wilson Byl, S. Erin Hannah, John L. Nitiss^¶, and Neil Osheroff

From the Departments of  Biochemistry and  Medicine (Oncology), Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0146 and the ^¶ Department of Molecular Pharmacology, St. Jude Children's Research Hospital, Memphis, Tennessee 38101

To further characterize the mechanistic basis for cellular resistance/hypersensitivity to anticancer drugs, a yeast genetic system was used to select a mutant type II topoisomerase that conferred cellular resistance to CP-115,953, amsacrine, etoposide, and ellipticine. The mutant enzyme contained a single point mutation that converted Gly⁴³⁷  Ser (top2G437S). Purified top2G437S displayed wild-type enzymatic activity in the absence of drugs but exhibited two properties that were not predicted by the cellular resistance phenotype. First, in the absence of ATP, it was hypersensitive to all of the drugs examined and hypersensitivity correlated with increased drug affinity. Second, in the presence of ATP, top2G437S lost its hypersensitivity and displayed wild-type drug sensitivity. Since the resistance of yeast harboring top2G437S could not be explained by alterations in enzyme-drug interactions, physiological levels of topoisomerase II were determined. The Gly⁴³⁷  Ser mutation reduced the stability of topoisomerase II and decreased the cellular concentration of the enzyme. These findings suggest that the physiological drug resistance phenotype conferred by top2G437S results primarily from its decreased stability. This study highlights the need to analyze both the biochemistry and the physiology of topoisomerase II mutants with altered drug sensitivity in order to define the mechanistic bridge that links enzyme function to cellular phenotype.

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