ABSTRACT2-Aminopurine (AP), a potent mutagenic base analogue, most frequently pairs with thymine. In the APT base pair, both bases adopt normal tautomeric forms. The mechanism for the mutagenic activity arises from its observed pairing with cytosine, which has been ascribed to an enhanced tendency to adopt the rare imino tautomeric form. NMR studies in H20 on all the exchangeable protons in an oligonucleotide duplex containing an APT base pair show WatsonCrick hydrogen bonding. When the thymine is replaced by cytosine in the duplex, we observe an AP-C base pair. Both amino protons of AP are seen excluding the rare tautomeric form. Although several alternative structures are possible, it is shown that the second hydrogen bond is formed by protonation of the AP-C base pair and that this is the dominant species under physiological conditions. bases remaining as favored amino tautomers. A further pairing possibility, prompted by recent NMR (11) and crystallographic data (12) on the T-G mismatch, would invoke a wobble base pair between AP and cytosine.We have synthesized two heteroduplex structures1 23 4 567 Studies using purified polymerases show that AP substitution for adenine in DNA results in a large increase in dCMP misincorporation (3). UV spectral and melting analyses of synthetic DNA polymers containing APRC base pairs (8) suggested that the mispair is stabilized by two hydrogen bonds, an expected 2-2 H bond between the amino group of AP and the keto oxygen of cytosine, and a shared 1-3 proton, which is not normally present when either base is present as the favored amino tautomer.Disfavored tautomers involving either AP or cytosine are only two among several possible structures for AP-C stabilized by two H bonds (Fig. 1). Indeed, theoretical calculations (9) indicate that AP is no more likely to form the rare tautomer than adenine or cytosine. Recent thermodynamic calculations (10, 21) suggest that the base pair between AP and cytosine may be stabilized by protonation with both EXPERIMENTALThe three oligonucleotides were synthesized as described (22).NMR Spectroscopy: Spectra were recorded at 500 MHz on a Bruker WM-500 at the Southern California Regional NMR facility at the California Institute of Technology (Pasadena, CA). The solvent peak was suppressed by a 1-t-1 hard pulse sequence (13). For most experiments, the carrier was placed at =11.5 ppm. For some spectra, it was placed much closer to water (see text).Oligonucleotides were 4 mM in strand concentration and were dissolved in 90% H20/10% 2H20/150 mM NaCl/10 mM phosphate, pH 7.2 (unless otherwise stated). Chemical shifts were reported relative to internal tetramethylammonium chloride at 3.18 ppm. RESULTSThe determination of the mode of base pairing between AP and cytosine depends on the identification and characterization of the exchangeable protons of the AP-C base pair. We first measured the spectra in H20 of a number of model compounds that relate to possible structures for the AP-C base pair.At pH 7.2, N3-methyl 2'-deoxycytidine (3 MedC), which i...

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