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Stability of the topoisomerase II closed clamp conformation may influence DNA-stimulated ATP hydrolysis

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Type II DNA topoisomerases catalyze changes in DNA topology and use nucleotide binding and hydrolysis to control conformational changes required for the enzyme reaction. We examined the ATP hydrolysis activity of a bisdioxopiperazine-resistant mutant of human topoisomerase II alpha with phenylalanine substituted for tyrosine at residue 50 in the ATP hydrolysis domain of the enzyme. This substitution reduced the DNA-dependent ATP hydrolysis activity of the mutant protein without affecting the relaxation activity of the enzyme. A similar but stronger effect was seen when the homologous mutation (Tyr28 --> Phe) was introduced in yeast Top2. The ATPase activities of human TOP2alpha(Tyr50 --> Phe) and yeast Top2(Tyr28 --> Phe) were resistant to both bisdioxopiperazines and the ATPase inhibitor sodium orthovanadate. Like bisdioxopiperazines, vanadate traps the enzyme in a salt-stable closed conformation termed the closed clamp, which can be detected in the presence of circular DNA substrates. Consistent with the vanadate-resistant ATPase activity, salt-stable closed clamps were not detected in reactions containing the yeast or human mutant protein, vanadate, and ATP. Similarly, ADP trapped wild-type topoisomerase II as a closed clamp, but could not trap either the human or yeast mutant enzymes. Our results demonstrate that bisdioxopiperazine-resistant mutants exhibit a difference in the stability of the closed clamp formed by the enzyme and that this difference in stability may lead to a loss of DNA-stimulated ATPase. We suggest that the DNA-stimulated ATPase of topoisomerase II is intimately connected with steps that occur while the N-terminal domain of the enzyme is dimerized.

Original languageEnglish
JournalThe journal of biological chemistry
Issue number12
Pages (from-to)11920-9
Number of pages10
Publication statusPublished - 25 Mar 2005

    Research areas

  • Adenosine Diphosphate/pharmacology, Adenosine Triphosphatases/metabolism, Adenosine Triphosphate/metabolism, Antigens, Neoplasm, DNA/pharmacology, DNA Topoisomerases, Type II/chemistry, DNA-Binding Proteins, Enzyme Stability, Humans, Hydrolysis, Poly-ADP-Ribose Binding Proteins, Protein Conformation, Razoxane/pharmacology, Vanadates/pharmacology

ID: 59178917