Dissecting the broad substrate specificity of human 3-methyladenine-DNA glycosylase

Human alkyladenine-DNA glycosylase (AAG) catalyzes the excision of a broad range of modified bases, protecting the genome from many types of alkylative and oxidative DNA damage. We have investigated how AAG discriminates against normal DNA bases, while accommodating a structurally diverse set of lesioned bases, by measuring the rates of AAG-catalyzed (k(st)) and spontaneous N-glycosidic bond hydrolysis (k(non)) for damaged and undamaged DNA oligonucleotides. The rate enhancements for excision of different bases reveal that AAG is most adept at excising the deaminated lesion hypoxanthine (k(st)/k(non) = 10(8)), suggesting that enzymatic activity may have evolved in response to this lesion. Comparisons of the rate enhancements for excision of normal and modified purine nucleobases provide evidence that AAG excludes the normal purines via steric clashes with the exocyclic amino groups of adenine and guanine. However, methylated purines are more chemically labile, and only modest rate enhancements are required for their efficient excision. Base flipping also contributes to specificity as destabilized mismatched base pairs are better substrates than stable Watson-Crick pairs, and many of the lesions recognized by AAG are compromised in their ability to base pair. These findings suggest that AAG reconciles a broad substrate tolerance with the biological imperative to avoid normal DNA by excluding normal bases from the active site rather than by specifically recognizing each lesion.