Solution structures of a duplex containing an adenine opposite a gap (absence of one nucleotide) - An NMR study and molecular dynamic simulations with explicit water molecules

We investigated the behaviour of a 15mer DNA duplex, [5 'd(CAGAGTCACTGGCTC) 3 ']. [5'd(GAGCCAG)3' + 5'd(GACTCTG)3'] which contained an adenine opposite the gap. Analysis of the NMR data showed the existence of one major specie s, which was in equilibrium with two minor species. Their relative concentr ations varied as a function of pH with a pK(a) of approximate to 4.5. For t he major species, the duplex was globally in B conformation with the centra l adenine stacked in the helix. The two G.C base pairs adjacent to the cent ral adenine were well formed and a gap was present in front of this adenine . For the minor species, major structural perturbations occurred in the cen tre of the duplex. At neutral pH, the central adenine was involved in a G.A mismatch with G23 adjacent to the gap. Cytosine C7 was then extrahelical a nd no gap was observed. Under these conditions, the major neutral species c orresponded to 70% of the total and the minor species to 30%. At acidic pH, the central adenine of the minor species was protonated and was involved i n a G(syn).A(+)(anti) mismatch. The difference is that C9 is now extrahelic al and G22 is implicated in the mispair. Three-dimensional models were buil t to initiate molecular dynamic simulations, which were in good agreement w ith the NMR data. Their structural stability in terms of hydrogen bonding a nd their flexibility are discussed and the biological significance for the interaction with DNA polymerase is evoked.