title: Characterization of a camptothecin-resistant human DNA topoisomerase I.
abstract: Topoisomerase I purified from a camptothecin-resistant human leukemia cell line and from the parental, camptothecin-sensitive line were compared in vitro. Relaxation of supercoiled DNA by the wild type enzyme was inhibited in the presence of camptothecin, while the mutant enzyme was unimpaired. Camptothecin altered the cleavage pattern of the wild type but not of the mutant enzyme. The stability of cleavable complexes was studied at a preferred topoisomerase I-binding sequence recognized by both enzymes. Camptothecin greatly enhanced the kinetic stability of the cleavable complex formed by the wild type enzyme, whereas that of the mutant enzyme was only marginally affected. In the absence of camptothecin, the cleavable complex formed by the mutant enzyme was stabilized relative to that of the wild type by several criteria. Thus, the mutant enzyme cleaved the topoisomerase I recognition sequence with 2-fold higher efficiency than the wild type enzyme. The mutant cleavable complex had a higher kinetic stability and was less sensitive to salt dissociation than the wild type complex. Furthermore, the mutant enzyme formed cleavable complexes in the absence of divalent cations, which were required for complex formation by the wild type enzyme.
authors.affiliation: Department of Molecular Biology and Plant Physiology, University of Aarhus, Denmark.
authors.forename: E
authors.initials: E
authors.lastname: Kjeldsen
authors.forename: B J
authors.initials: BJ
authors.lastname: Bonven
authors.forename: T
authors.initials: T
authors.lastname: Andoh
authors.forename: K
authors.initials: K
authors.lastname: Ishii
authors.forename: K
authors.initials: K
authors.lastname: Okada
authors.forename: L
authors.initials: L
authors.lastname: Bolund
authors.forename: O
authors.initials: O
authors.lastname: Westergaard
mesh_terms: D001483:Base Sequence; D002166:Camptothecin / Q000494:pharmacology*; D002460:Cell Line; D004264:DNA Topoisomerases, Type I / Q000378:metabolism*; D004351:Drug Resistance; D006801:Humans; D007700:Kinetics; D007945:Leukemia, Lymphoid; D013379:Substrate Specificity
chemical_list: D004264:DNA Topoisomerases, Type I; D002166:Camptothecin