SOLUTION STRUCTURE OF 2 MISMATCHES A-CENTER-DOT-A AND T-CENTER-DOT-T IN THE K-RAS GENE CONTEXT BY NUCLEAR-MAGNETIC-RESONANCE AND MOLECULAR-DYNAMICS

Two mismatches, one homopurine (A . A) and the other homopyrimidine (T . T), have been incorporated at the central position N of: 5'd(GCCACN AGCTC) . d(GAGCTNGTGGC) in order to study nuclear magnetic resonance s pectra and molecular dynamics. These duplexes constitute the sequence 29-39 of the K-ras gene coding for Gly12, a hot spot for mutation. The NMR spectra show that the duplexes are not greatly distorted by the i ntroduction of the mismatches and their global conformation is that of a canonical B-form double helix. For both systems, no structural chan ge is observed in the pH range 4.7-9. For the duplex containing the ho mopurine A . A mismatch, we propose a type of pairing involving one hy drogen bond between the amino group of one central adenine and the nit rogen N1 of the opposite adenine. For the duplex containing the mispai red T . T bases, NMR spectra recorded in H2O at 282 K indicate that th ese central bases are engaged in wobble pairing, involving two imino-c arbonyl hydrogen bonds. For both systems two conformations with the sa me donor and acceptor pattern can coexist, one being obtained from the other by a 180 degrees rotation about the pseudodyadic axis. Exchange between the two forms is observed by NMR at low temperature for the T . T mispair and also inferred from NMR measurements on the A . A syst em. The presence of this exchange and its pathway has been investigate d by molecular dynamics calculations on both systems. Distance restrai ned and unrestrained molecular dynamics are in very good agreement wit h the NMR data. The average structure for either mispair shows only sm all conformational change from normal B DNA. For each, a systematic pa thway is observed for exchange between the two conformations.