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

Thomas Spratt, Colin R. Campbell

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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 SN2 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.

Original languageEnglish (US)
Pages (from-to)11364-11371
Number of pages8
Issue number37
StatePublished - Sep 1 1994

All Science Journal Classification (ASJC) codes

  • Biochemistry


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