Studies on the Catalytic Mechanism of Five DNA Glycosylases

Abstract

DNA glycosylases catalyze scission of the N-glycosylic bond linking a damaged base to the DNA sugar phos- phate backbone. Some of these enzymes carry out a con- comitant abasic (apyrimidinic/apurinic (AP)) lyase reac- tion at a rate approximately equal to that of the glycosylase step. As a generalization of the mechanism described for T4 endonuclease V, a repair glycosy- lase/AP lyase that is specific for ultraviolet light-in- duced cis-syn pyrimidine dimers, a hypothesis concern- ing the mechanism of these repair glycosylases has been proposed. This hypothesis describes the initial action of all DNA glycosylases as a nucleophilic attack at the sugar C-1' of the damaged base nucleoside, resulting in scission of the N-glycosylic bond. It is proposed that the enzymes that are only glycosylases differ in the chemi- cal nature of the attacking nucleophile from the glyco- sylase/AP lyases. Those DNA glycosylases, which carry out the AP lyase reaction at a rate approximately equal to the glycosylase step, are proposed to use an amino group as the nucleophile, resulting in an imino enzyme- DNA intermediate. The simple glycosylases, lacking the concomitant AP lyase activity, are proposed to use some nucleophile from the medium, e.g. an activated water molecule. This paper reports experimental tests of this hypothesis using five representative enzymes, and these data are consistent with this hypothesis.