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.
Highlights
From the Department of Molecular Biology, University of Aarhus, C
Mechanism of Hydrolysis and Alcoholysis Mediated by Eukaryotic Topoisomerase I-’h. characterize the catalytic repertoire of eukaryotic topoisomerase I, we have searched for conditions in which the covalent enzyme-DNAcomplexes can mediate hydrolysis of the 3‘-phosphotyrosylbond
Miscellaneous short chain polyhydric alcoholcompounds function as nucleophiles during the release of topoisomerase I a t pH 9
Summary
Reagents and Enzymes-Ethyleneglycol and glycerol were from Merck. meso-Erythritol,adonitol, xylitol, L-arabitol,0-arabitol, dulcitol, D-mannitol, D-glucitol, MES,' Tris, CHES, and CAPS were from Sigma.
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