STUDIES ON THE CATALYTIC MECHANISM OF 5 DNA GLYCOSYLASES - PROBING FOR ENZYME-DNA IMINO INTERMEDIATES

DNA glycosylases catalyze scission of the N-glycosylic bond linking a damaged base to the DNA sugar phosphate backbone, Some of these enzyme s carry out a concomitant abasic (apyrimidinic/apurinic (AP)) lyase re action at a rate approximately equal to that of the glycosylase step. As a generalization of the mechanism described for T4 endonuclease V, a repair glycosylase/AP lyase that is specific for ultraviolet light-i nduced cis-syn pyrimidine dimers, a hypothesis concerning the mechanis m of these repair glycosylases has been proposed, This hypothesis desc ribes the initial action of all DNA glycosylases as a nucleophilic att ack at the sugar C-1' of the damaged base nucleoside, resulting in sci ssion of the N-glycosylic bond. It is proposed that the enzymes that a re only glycosylases differ in the chemical nature of the attacking nu cleophile from the glycosylase/AP lyases. Those DNA glycosylases, whic h carry out the AP lyase reaction at a rate approximately equal to the glycosylase step, are proposed to use an amino group as the nucleophi le, resulting in an imino enzyme DNA intermediate. The simple glycosyl ases, lacking the concomitant AP lyase activity, are proposed to use s ome nucleophile from the medium, e.g. an activated water molecule, Thi s paper reports experimental tests of this hypothesis using five repre sentative enzymes, and these data are consistent with this hypothesis.