Alteration of A•T base-pair opening kinetics by the ammonium cation in DNA A-tracts

We have investigated by NMR the effects of NH4+ on the chemical shifts, on the structure, and on the imino proton exchange kinetics of two duplexes containing an A-tract, [d(CGCGAATTCGCG)](2) and [d(GCA(4)T(4)GC)](2), and of a B-DNA duplex, [d(CGCGATCGCG)](2). Upon NH4+ addition to [d(CGCGAATTCGCG)](2), the adenosine H-2 protons, the thymidine imino protons, and the guanosine imino proton of the adjacent G.C pair show unambiguous chemical shifts. Similar shifts are observed in the A-tract of [d(GCA(4)T(4)GC)](2) and for the A5(H2) proton of the B DNA duplex [d(CGCGATCGCG)](2). The localization of the shifted protons suggests an effect related to NH4+ binding in the minor groove. The cross-peak intensities of the NOESY spectra collected at low and high NH4+ concentrations are comparable, and the COSY spectra do not show any change of the sugar pucker. This indicates a modest effect of ammonium binding on the duplex structures. Nevertheless, the imino proton exchange catalysis by ammonia provides evidence for a substantial effect of NH4+ binding on the A.T base-pair kinetics in the A-tracts. Proton exchange experiments performed at high and low NH4+ concentrations show the occurrence of two native conformations in proportions depending on the NH4+ concentration. The base-pair lifetimes and the open-state lifetimes of each conformation are distinct. Exchange from each conformation proceeds via a single open state. But if, and only if, the NH4+ concentration is kept larger than I M, the A.T imino proton exchange times of A-tract sequences exhibit a linear dependence versus the inverse of the NH3 proton acceptor concentration. This had been interpreted as an indication for two distinct base-pair opening modes (Warmlander, S., Sen, A., and Leijon, M. (2000) Biochemistry 39, 607-615).