Multiple structures for the 2-aminopurine-cytosine mispair

The base pair formed between 2-aminopurine (2AP) and cytosine (C) is an intermediate in transition mutations generated by 2AP. To date, several structures have been proposed for the 2AP-C mispair, including those involving a rare tautomer, a protonated base pair, and a neutral wobble structure. In this paper, we describe a series of UV, fluorescence, and NMR studies which demonstrate that an equilibrium exists between the neutral wobble and the protonated Watson-Crick structures. The apparent pK value for the transition between the structures is 5.9-6.0. Formation of a Watson-Crick base pair is accomplished predominantly by protonation of the 2AP residue as indicated by UV spectral changes, fluorescence quenching, and changes in proton chemical shifts. Rapid transfer of the shared proton between the 2AP and cytosine residues is indicated by the rapid exchange of the cytosine amino protons of the protonated Watson-Crick configuration. The relative contribution of the neutral wobble and protonated Watson-Crick configurations to 2AP-induced transition mutations is discussed.