Characterization of an altered DNA catalysis of a camptothecin-resistant eukaryotic topoisomerase I.
We investigated topoisomerase I activity at a specific camptothecin-enhanced cleavage site by use of a partly double-stranded DNA substrate. The cleavage site belongs to a group of DNA topoisomerase I sites which is only efficiently cleaved by wild-type topoisomerase I (topo I-wt) in the presence of camptothecin. With a mutated camptothecin-resistant form of topoisomerase I (topo I-K5) previous attempts to reveal cleavage activity at this site have failed. On this basis it was questioned whether the mutant enzyme has an altered DNA sequence recognition or a changed rate of catalysis at the site. Utilizing a newly developed assay system we demonstrate that topo I-K5 not only recognizes and binds to the strongly camptothecin-enhanced cleavage site but also has considerable cleavage/religation activity at this particular DNA site. Thus, topo I-K5 has a 10-fold higher rate of catalysis and a 10-fold higher affinity for DNA relative to topo I-wt. Our data indicate that the higher cleavage/religation activity of topo I-K5 is a result of improved DNA binding and a concomitant shift in the equilibrium between cleavage and religation towards the religation step. Thus, a recently identified point mutation which characterizes the camptothecin-resistant topo I-K5 has altered the enzymatic catalysis without disturbing the DNA sequence specificity of the enzyme.
Department of Molecular Biology, University of Aarhus, Denmark.
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Gromova
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Kjeldsen
J Q
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Svejstrup
J
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Alsner
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Christiansen
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Westergaard
D001483:Base Sequence; D002166:Camptothecin / Q000494:pharmacology*; D002384:Catalysis; D004247:DNA / Q000378:metabolism*; D004264:DNA Topoisomerases, Type I / Q000187:drug effects / Q000378:metabolism*; D004351:Drug Resistance; D006801:Humans; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides / Q000138:chemical synthesis / Q000378:metabolism; D013997:Time Factors; D014407:Tumor Cells, Cultured
D009838:Oligodeoxyribonucleotides; D004247:DNA; D004264:DNA Topoisomerases, Type I; D002166:Camptothecin