SOLUTION STRUCTURE OF AN ONCOGENIC DNA DUPLEX, THE K-RAS GENE AND THESEQUENCE CONTAINING A CENTRAL C-CENTER-DOT-A OR A-CENTER-DOT-G MISMATCH AS A FUNCTION OF PH - NUCLEAR-MAGNETIC-RESONANCE AND MOLECULAR-DYNAMICS STUDIES

The DNA duplex 5' d(GCCACCAGCTC)-d(GAGCTGGTGGC) corresponds to the seq uence 29 to 39 of the K-ms gene, which contains a hot spot for mutatio ns. This has been studied by one and two-dimensional nuclear magnetic resonance, energy minimization and molecular dynamics. The results sho w that it adopts a globally B-DNA type structure. We have introduced, at the central base-pair, the mismatches C.A and A.G. The mismatch pos ition is that of the first base of the Gly12 codon, the hot spot. For the C.A mismatch we observe a structural change as a function of pH wi th an apparent pK(a) of 7.2. At low pH, the mismatch pair adopts a str ucture close to a classic wobble conformation with the cytidine residu e displaced into the major groove. It is stabilised by two hydrogen bo nds in which the adenosine residue is protonated and the cytidine resi due has a significant C3'-endo population. At high pH, the mispair str ucture is in equilibrium between wobble and reverse wobble conformatio ns. Similar studies are reported on the A.G mismatch, which also under goes a transition as a function of pH. P-31 spectra have been recorded on all systems and as a function of pH. No evidence for B-II phosphod iester backbone conformations was found. The NMR results are well corr oborated by molecular dynamics calculations performed with or without distance constraints. The dynamics at the mismatch sites have been exa mined. Although the overall structures are close to B-DNA, helical par ameters fluctuate differently at these sites. Different hydrogen bondi ng alternatives in dynamic equilibrium that can involve three-centred hydrogen bonds are observed.