Active-site clashes prevent the human 3-methyladenine DNA glycosylase from improperly removing bases

The human 3-methyladenine DNA glycosylase (AAG, MPG) removes a diverse array of damaged purines via a nucleotide-flipping mechanism. In the crystal structure of AAG bound to DNA containing 1,N-6 ethenoadenine, an asparagine (N169) occupies the active-site floor, in close proximity to the C-2 position of the flipped-out 1,N-6 ethenoadenine. We engineered site-specific AAG mutants to determine whether N169 prevents normal bases from mistakenly entering the active site. Substituting alanine or serine resulted in mutants that excised substrates at a faster rate than wild-type. Furthermore, these mutants acquired the ability to excise normal guanine within mispairs but not opposite cytosine. The results suggest that AAG can recognize helical deformations, such as mispairs. However, the active site then prevents the mistaken excision of bases, which prevents AAG from acquiring a mutator activity.