title: The covalent eukaryotic topoisomerase I-DNA intermediate catalyzes pH-dependent hydrolysis and alcoholysis.
abstract: Eukaryotic topoisomerase I catalysis was characterized by the use of a DNA substrate system, which allows uncoupling of cleavage and ligation half-reactions. Covalent topoisomerase I-DNA intermediates formed by cleavage without concomitant ligation were able to catalyze hydrolysis of the 3'-phosphotyrosyl bond in the pH range 7.5-10, with a broad pH optimum between pH 8.5 and 9.5. In comparison, the DNA cleavage and ligation activity of topoisomerase I were found to be independent of pH in the pH range 7-10 and strongly impaired at higher pH values. Moreover, different polyhydric alcohol compounds were found to function as nucleophiles at pH 9 to facilitate the release of topoisomerase I. The hydrolysis and alcoholysis activities of topoisomerase I were specific for the 3'-phosphotyrosyl bond and blocked by enzyme denaturation or proteolysis. Taken together the data suggest that site-specific hydrolysis or alcoholysis mediated by topoisomerase I-DNA complexes reflects the ability of the enzyme to direct the activation of the 3'-phosphotyrosyl bond or the incoming nucleophile. Analysis of enzyme-directed coupling of non-DNA nucleophiles to the cleaved DNA strand may provide a useful tool for elucidation of the biochemical mechanism of type I DNA topoisomerases.
authors.affiliation: Department of Molecular Biology, University of Aarhus, Denmark.
authors.forename: K
authors.initials: K
authors.lastname: Christiansen
authors.forename: B R
authors.initials: BR
authors.lastname: Knudsen
authors.forename: O
authors.initials: O
authors.lastname: Westergaard
mesh_terms: D001483:Base Sequence; D002384:Catalysis; D003001:Cloning, Molecular; D004247:DNA / Q000378:metabolism*; D004264:DNA Topoisomerases, Type I / Q000302:isolation & purification / Q000378:metabolism*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007700:Kinetics; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides / Q000138:chemical synthesis / Q000737:chemistry / Q000378:metabolism*; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D012441:Saccharomyces cerevisiae; D013379:Substrate Specificity
chemical_list: D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; D004247:DNA; D004264:DNA Topoisomerases, Type I