Cytosine methylation effects on the repair of O⁶-methylguanines within CG dinucleotides

O⁶-Alkyldeoxyguanine adducts induced by tobacco-specific nitrosamines are repaired by O⁶-alkylguanine DNA alkyltransferase (AGT), which transfers the O⁶-alkyl group from the damaged base to a cysteine residue within the protein. In the present study, a mass spectrometry-based approach was used to analyze the effects of cytosine methylation on the kinetics of AGT repair of O⁶-methyldeoxyguanosine (O⁶-Me-dG) adducts placed within frequently mutated 5′-CG-3′ dinucleotides of the p53 tumor suppressor gene. O⁶-Me-dG-containing DNA duplexes were incubated with human recombinant AGT protein, followed by rapid quenching, acid hydrolysis, and isotope dilution high pressure liquid chromatography-electrospray ionization tandem mass spectrometry analysis of unrepaired O⁶-methylguanine. Second-order rate constants were calculated in the absence or presence of the C-5 methyl group at neighboring cytosine residues. We found that the kinetics of AGT-mediated repair of O⁶-Me-dG were affected by neighboring 5-methylcytosine (^MeC) in a sequence-dependent manner. AGT repair of O⁶-Me-dG adducts placed within 5′-CG-3′ dinucleotides of p53 codons 245 and 248 was hindered when ^MeC was present in both DNA strands. In contrast, cytosine methylation within p53 codon 158 slightly increased the rate of O⁶-Me-dG repair by AGT. The effects of ^MeC located immediately 5′ and in the base paired position to O⁶-Me-dG were not additive as revealed by experiments with hypomethylated sequences. Furthermore, differences in dealkylation rates did not correlate with AGT protein affinity for cytosine-methylated and unmethylated DNA duplexes or with the rates of AGT-mediated nucleotide flipping, suggesting that ^MeC influences other kinetic steps involved in repair, e.g. the rate of alkyl transfer from DNA to AGT.