Efficient recognition of substrates and substrate analogs by the adenine glycosylase MutY requires the C-terminal domain

The Escherichia coil DNA repair enzyme MutY plays an important role in the prevention of DNA mutations by removing misincorporated adenine residues fr om 7,8-dihydro-8-oxo-2'-deoxyguanosine:2'-deoxyadenosine (OG:A) mispairs, T he N-terminal domain of MutY (Stop 225, Met1-Lys225) has a sequence and str ucture that is characteristic of a superfamily of base excision repair glyc osylases; however, MutY and its homologs contain a unique C-terminal domain . Previous studies have shown that the C-terminal domain confers specificit y for OG:A substrates over G:A substrates and exhibits homology to the d(OG )TPase MutT, suggesting a role in OG recognition. In order to provide addit ional information on the importance of the C-terminal domain in damage reco gnition, we have investigated the kinetic properties of a form lacking this domain (Stop 225) under multiple- and single-turnover conditions, In addit ion, the interaction of Stop 225 with a series of non-cleavable substrate a nd product analogs was evaluated using gel retardation assays and footprint ing experiments, Under multiple-turnover conditions Stop 225 exhibits bipha sic kinetic behavior with both OG:A and G:A substrates, likely due to rate- limiting DNA product release. However, the rate of turnover of Stop 225 was increased a-fold with OG:A substrates compared to the wild-type enzyme. In contrast, the intrinsic rate for adenine removal by Stop 225 from both G:A and OG:A substrates is significantly reduced (10- to 25-fold) compared to the wild-type. The affinity of Stop 225 for substrate analogs was dramatica lly reduced, as was the ability to discriminate between substrate analogs p aired with OG over G, Interestingly, similar hydroxyl radical and DMS footp rinting patterns are observed for Stop 225 and wild-type MutY bound to DNA duplexes containing OG opposite an abasic site mimic or a non-hydrogen bond ing A analog, suggesting that similar regions of the DNA are contacted by b oth enzyme forms. Importantly, Stop 225 has a reduced ability to prevent DN A mutations in vivo. This implies that the reduced adenine glycosylase acti vity translates to a reduced capacity of Stop 225 to prevent DNA mutations in vivo.