Interactions of the human, rat, Saccharomyces cerevisiae and Escherichia coli 3-methyladenine-DNA glycosylases with DNA containing dIMP residues

Abstract

In DNA, the deamination of dAMP generates 2'-deoxyinosine 5'-monophosphate (dIMP). Hypoxanthine (HX) residues are mutagenic since they give rise to A·T→G·C transition. They are excised, although with different efficiencies, by an activity of the 3-methyladenine (3-meAde)-DNA glycosylases from Escherichia coli (AlkA protein), human cells (ANPG protein), rat cells (APDG protein) and yeast (MAG protein). Comparison of the kinetic constants for the excision of HX residues by the four enzymes shows that the E. coli and yeast enzymes are quite inefficient, whereas for the ANPG and the APDG proteins they repair the HX residues with an efficiency comparable to that of alkylated bases, which are believed to be the primary substrates of these DNA glycosylases. Since the use of various substrates to monitor the activity of HX-DNA glycosylases has generated conflicting results, the efficacy of the four 3-meAde-DNA glycosylases of different origin was compared using three different substrates. Moreover, using oligonucleotides containing a single dIMP residue, we investigated a putative sequence specificity of the enzymes involving the bases next to the HX residue. We found up to 2-5-fold difference in the rates of HX excision between the various sequences of the oligonucleotides studied. When the dIMP residue was placed opposite to each of the four bases, a preferential recognition of dl:T over dl:dG, dl:dC and dl:dA mismatches was observed for both human (ANPG) and E. coli (AlkA) proteins. At variance, the yeast MAG protein removed more efficiently HX from a dl:dG over dl:dC, dl:T and dl:dA mismatches.