Structural Basis for Nucleobase Activation by the Adenine DNA Glycosylase MutY

The DNA glycosylase MutY excises adenine when mispaired with oxidized guanine (OG). While it is understood that inappropriate adenine excision would be catastrophic, the mechanism by which MutY activity is kept in check and only licensed at OG:A lesions is unknown. To explore the structural basis for nucleobase activation, we tested kinetic and structural consequences following replacement of the catalytic Glu, a signature residue for MutY. E43Q and E43S substitution variants of MutY from Geobacillus stearothermophilus, though severely impaired, retained measurable activity. X-ray crystal structures showed the substrate nucleobase in an anti conformation, rotated by 180° from the syn conformation seen in previous substrate complexes. Remarkably, the AP product was observed as the alpha-anomer configuration when generated by these Glu-replacement variants, completely different from the beta-anomer AP product expected for the wild-type enzyme and seen directly for other cancer-associated variants. Our results suggest a mechanism for regulating MutY, whereby Glu engagement with the syn conformation of the nucleobase licenses a "go ahead" state for adenine excision only at OG:A lesions, while Glu dis-engagement establishes an "on hold" state to avoid inappropriate activity elsewhere.