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'-deoxy-inosine 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-methyl-adenine (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 oligo-nucleotides 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 dI:T over dI:dG, dI:dC and dI: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 dI:dG over dI:dC, dI:T and dI:dA mismatches.

Figure 1

Activity of the ANPG40 and AlkA proteins on duplex deoxyribonucleotides containing dIMP residues. The ³²P-5′-end labeled double-stranded oligonucleotide HX20-34/T, after various treatments, was analyzed by PAGE under denaturing conditions. Lane 1, no treatment; lane 2, treated with piperidine (1 M, 30 min, 90°C); lane 3, treated with the Fpg protein (100 ng, 20 min, 37°C); lanes 4–8, HX20-34/T was treated with the ANPG40 protein (0.5 U, 20 min, 37°C); lanes 10–14, treated with the AlkA protein (5 U, 60 min, 37°C). These samples were then further treated (lanes 5 and 11) either with piperidine (1 M, 30 min, 90°C) or with abasic site nicking enzymes (30 min at 37°C). Lanes 6 and 12, with the Fpg protein (100 ng); lanes 7 and 13, with the Nfo protein (500 ng); lanes 8 and 14, with the Nth protein (100 ng). Lane 9, the ³²P-5′-end labeled single-stranded oligonucleotide HX20-34/T was treated with the ANPG40 and Fpg proteins as above. Arrow a indicates the 34mer oligonucleotide. Arrow b points to the 19mer oligonucleotide with an α–β unsaturated aldehyde at the 3′-terminus. Arrow c points to the 19mer oligonucleotide with 3′-OH group at the 3′-terminus. Arrow d points to the 19mer oligonucleotide with a phosphate group at the 3′-terminus. For details see Materials and Methods.

Figure 2

Kinetics of excision, by the ANPG40 protein, of HX from an oligonucleotide duplex containing thymine opposite a deoxyinosine residue. (A) The oligonucleotide HX20-34/T (10 nM) was incubated with the ANPG40 protein (0.2 U) for increasing periods of time in the presence of Fpg protein. The products of the reaction were separated by electrophoresis on a 20% polyacrylamide gel containing 7 M urea and visualized. Lanes 1–7, incubation time 0, 5, 10, 15, 20, 25 and 30 min, respectively. (B) The radioactivity of the products of the reaction shown in (A) was quantified and plotted as a function of time. For details see Materials and Methods.

Figure 2

Kinetics of excision, by the ANPG40 protein, of HX from an oligonucleotide duplex containing thymine opposite a deoxyinosine residue. (A) The oligonucleotide HX20-34/T (10 nM) was incubated with the ANPG40 protein (0.2 U) for increasing periods of time in the presence of Fpg protein. The products of the reaction were separated by electrophoresis on a 20% polyacrylamide gel containing 7 M urea and visualized. Lanes 1–7, incubation time 0, 5, 10, 15, 20, 25 and 30 min, respectively. (B) The radioactivity of the products of the reaction shown in (A) was quantified and plotted as a function of time. For details see Materials and Methods.