EVIDENCE THAT MUTY IS A MONOFUNCTIONAL GLYCOSYLASE CAPABLE OF FORMINGA COVALENT SCHIFF-BASE INTERMEDIATE WITH SUBSTRATE DNA

The Escherichia coli adenine glycosylase MutY is involved in the repai r of 7,8-dihydro-8-oxo-2'-deoxyguanosine (OG):A and G:A mispairs in DN A, DNA strand cleavage via beta-elimination (beta-lyase) activity coup led with MutY's removal of misincorporated adenine bases was sought us ing both qualitative and quantitative methods. The qualitative assays demonstrate formation of a Schiff base intermediate which is character istic of DNA glycosylases catalyzing a concomitant beta-lyase reaction . Borohydride reduction of the Schiff base results in the formation of a covalent DNA-MutY adduct which is easily detected in SDS-PAGE exper iments. However, quantitative activity assays which monitor DNA strand scission accompanying base release suggest MutY behaves as a simple m onofunctional glycosylase. Treatment with base effects DNA strand clea vage at apurinic/apyrimidinic (AP) sites arising via simple glycosylas e activity. The amount of cleaved DNA in MutY reactions treated with b ase is much greater than that in non-base treated reactions, indicatin g that AP site generation by MutY is not associated with a concomitant beta-lyase step. As standards, identical assays were performed with a known monofunctional enzyme (uracil DNA glycosylase) and a known bifu nctional glycosylase/lyase (FPG), the results of which were used in co mparison with those of the MutY experiments. The apparent inconsistenc y between the data obtained for MutY by the qualitative and quantitati ve methods underscores the current debate surrounding the catalytic ac tivity of this enzyme, and a detailed explanation of this controversy is proposed. The work presented here lays ground for the identificatio n of specific active site residues responsible for the chemical mechan ism of MutY enzyme catalysis.