Synthesis of Oligodeoxynucleotides Containing Analogs of O⁶-Methylguanine and Reaction with O⁶-Alkylguanine-DNA Alkyltransferase

O⁶-Alkylguanine-DNA alkyltransferase (AGT) repairs the mutagenic O⁶-methylguanine (O⁶-mG) lesion by transferring a methyl group from the 6-position of guanine to a cysteine residue on the protein. The simplest possible mechanism is an S_N2 process in which the cysteine displaces the methyl group off of the guanine in a concerted reaction. To probe the interactions between the protein and guanine leaving group, oligodeoxynucleotide duplexes containing analogs of O⁶mG were synthesized and then reacted with AGT. The analogs, which were incorporated into deoxynucleotides include O⁶-methylhypoxanthine (O⁶-mH),S⁶-methyl-6-thioguanine (S⁶mG),S⁶-methyl-6-thiohypoxanthine (S⁶mH),Se⁶-methyl-6-selenoguanine (Se⁶mG),Se⁶-methyl-6-selenohypoxanthine (Se⁶mH), O⁶-methy1-1 -deazaguanine (O⁶mlDG), O⁶-methyl-3-deazaguanine (O⁶m3DG), and O⁶-methyl-7-deazaguanine (O⁶m7DG), differ from O⁶mG in that the heteroatoms have been replaced so that they are poorer hydrogen bond participants and proton acceptors. AGT was reacted with oligonucleotide duplexes of the sequence 5′-GGC GCT XGA GGC GTG-3′ in which X was O⁶mG or an analog in which X was paired with C. The reactions in 50 mM Tris-HCl and 1 mM EDTA, pH 7.6 and 37 °C, were followed by anion-exchange HPLC in 10 mM NaOH with a NaCl gradient. All detected reactions were demethylations of the oligodeoxynucleotides except for O⁶m3DG, which reacted in an unknown manner. The second-order rate constants obtained are as follows (M⁻¹ s⁻¹): O⁶mG, (7.7 ± 0.9) × 10⁵; O⁶mH, (5.4 ± 0.7) × 10⁶; S⁶mG, (1.7 ± 0.2) × 10³; S⁶mH, not detected; Se⁶mG, (4.1 ± 1.6) × 10³; Se⁶mH, not detected; O⁶mlDG, (2.2 ± 0.9) × 10³; O⁶m3DG, (7.7 ± 2.5) × 10⁴; O⁶m7DG, (3.1 ± 1.0) × 10⁴. The large decreases in rate observed for changing the oxygen at the 6-position and the ring nitrogen at the 1-position suggest that these sites are hydrogen bond acceptors and/or proton acceptors during the reaction. The potential hydrogen bond from the protein to the 1-position of O⁶mG as well as the increase in rate observed for O⁶mH suggests that the duplex opens up in order for the reaction to occur.