H-1-NMR OF AN OLIGODEOXYNUCLEOTIDE CONTAINING A PROPANODEOXYGUANOSINEADDUCT POSITIONED IN A (CG)(3) FRAMESHIFT HOTSPOT OF SALMONELLA-TYPHIMURIUM HISD3052 - HOOGSTEEN BASE-PAIRING AT PH-5.8

The exocyclic DNA adduct 1,N-2-propano-2'-deoxyguanosine (PdG) was ins erted into the oligodeoxynucleotide 5'-CGC(PdG)CGGCATG-3' and annealed to the complementary oligodeoxynucleotide 5'-CATGCCGCGCG-3'. This seq uence is derived from a spontaneous revertant of the hisD3052 gene in a frameshift-sensitive tester strain of Salmonealla typhimurium and is a hotspot for two-base pair deletions. The solution structure of the modified duplex was examined by H-1 NMR spectroscopy. The exocyclic le sion resulted in loss of Watson-Crick base-pairing capability. Modific ation resulted in an similar to 24 degrees C decrease in T-m of the du plex. NMR experiments revealed pH-dependent conformational equilibria, which involved the modified base pair and its 3'-neighbor base pair. At pH 5.8, the lesion resulted in a localized perturbation of the B-fo rm helix. PdG was rotated about the glycosyl bond from the anti to the syn conformation, thus placing the propane moiety into the major groo ve. This resulted in the observation of a strong NOE between the imida zole proton of PdG and the anomeric proton of the attached deoxyribose . Additional NOEs were observed between the methylene protons of the p ropane moiety and H5 and H6 of the 5'-neighbor cytosine. An imino prot on resonance from the cytosine complementary to PdG and protonated at N3, characteristic of a Hoogsteen base pair, was observed at 15 ppm, b ut was broadened due to exchange with water. The amino protons of the complementary cytosine were shifted downfield from the other cytosine amino protons, characteristic of a Hoogsteen-like conformation at the site of modification. A second equilibrium involved the 3'-neighbor ba se pair, which alternated between Watson-Crick and Hoogsteen pairing, also via rotation of the guanosine glycosyl bond from the anti to the syn conformer. The conformational exchange of the 3'-neighbor base pai r was sufficiently slow on the NMR time scale to allow simultaneous ob servation of resonances from the Watson-Crick and the Hoogsteen confor mers.