1H NMR of an oligodeoxynucleotide containing a propanodeoxyguanosine adduct positioned in a (CG)3 frameshift hotspot of Salmonella typhimurium hisD3052: Hoogsteen base-pairing at pH 5.8.

The exocyclic DNA adduct 1,N2-propano-2'-deoxyguanosine (PdG) was inserted into the oligodeoxynucleotide 5'-CGC(PdG)CGGCATG-3' and annealed to the complementary oligodeoxynucleotide 5'-CATGCCGCGCG-3'. This sequence is derived from a spontaneous revertant of the hisD3052 gene in a frameshift-sensitive tester strain of Salmonella typhimurium and is a hotspot for two-base pair deletions. The solution structure of the modified duplex was examined by 1H NMR spectroscopy. The exocyclic lesions resulted in loss of Watson-Crick base-pairing capability. Modification resulted in an approximately 24 degrees C decrease in Tm of the duplex. 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-form helix. PdG was rotated about the glycosyl bond from the anti to the syn conformation, thus placing the propano moiety into the major groove. This resulted in the observation of a strong NOE between the imidazole proton of PdG and the anomeric proton of the attached deoxyribose. Additional NOEs were observed between the methylene protons of the propano moiety and H5 and H6 of the 5'-neighbor cytosine. An imino proton resonance from the cytosine complementary to PdG and protonated at N3, characteristic of a Hoogsteen base pair, was observed at 15 ppm, but 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 base 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 pair was sufficiently slow on the NMR time scale to allow simultaneous observation of resonances from the Watson-Crick and the Hoogsteen conformers.

Structure of a duplex oligodeoxynucleotide containing propanodeoxyguanosine opposite a two-base deletion in the (CpG)3 frame shift hotspot of Salmonella typhimurium hisD3052 determined by 1H NMR and restrained molecular dynamics.

Structural refinement from solution 1H NMR data was performed on the 5'-d[ATCGC(PdG)-CGGCATG]-3'.5'-d[CATGCCGCGAT]-3' duplex, in which the adducted oligodeoxynucleotide containing the exocyclic lesion 1,N2-propano-2'-deoxyguanosine (PdG) was annealed with the complementary strand which contained a CpG deletion. The resulting duplex required PdG and one adjacent cytosine to be unpaired. A total of 352 distances were utilized to restrain molecular dynamics calculations, of which 264 were NOE-derived. These distances were calculated using complete relaxation matrix methods from hybrid matrices, which were comprised of the experimentally determined distances and additional distances derived from either A-form or B-form DNA. A simulated annealing protocol combined with the distance restraints was able to refine a single structure with an average rms deviation of < 1.35 A. The accuracy of the refined structure was assessed using full relaxation matrix calculations, which gave good agreement with measured NOE intensities. PdG was found to be stacked into the helix below base pair C3.G18, whereas C5 was found to be unpaired and extruded toward the major groove and parallel to base pair G6.C17. This created a localized bend in the DNA helix of approximately 20-35 degrees at the junction between PdG and C5. The bending corroborated previous assays performed on this modified sequence [Moe, J. G., Reddy, G. R., Marnett, L. J., & Stone, M. P. (1994) Chem. Res. Toxicol. 7, 319-328].

Duplex DNA catalyzes the chemical rearrangement of a malondialdehyde deoxyguanosine adduct.

The primary DNA lesion induced by malondialdehyde, a byproduct of lipid peroxidation and prostaglandin synthesis, is 3-(2'-deoxy-beta-D-erythro-pentofuranosyl)-pyrimido[1, 2-a]purin-10(3H)-one (M1G). When placed opposite cytosine (underlined) at neutral pH in either the d(GGTMTCCG).d(CGGACACC) or d(ATCGCMCGGCATG). d(CATGCCGCGCGAT) duplexes, M1G spontaneously and quantitatively converts to the ring-opened derivative N2-(3-oxo-1-propenyl)-dG. Ring-opening is reversible on thermal denaturation. Ring-opening does not occur at neutral pH in single-stranded oligodeoxynucleotides or when T is placed opposite to M1G in a duplex. The presence of a complementary cytosine is not required to stabilize N2-(3-oxo-1-propenyl)-dG in duplex DNA at neutral pH. When N2-(3-oxo-1-propenyl)-dG is placed opposite to thymine in a duplex, it does not revert to M1G. A mechanism for the conversion of M1G to N2-(3-oxo-1-propenyl)-dG is proposed in which the exocyclic amino group of the complementary cytosine attacks the C8 position of the M1G exocyclic ring and facilitates ring opening via formation of a transient Schiff base. Addition of water to the Schiff base regenerates the catalytic cytosine and generates N2-(3-oxo-1-propenyl)-dG. These results document the ability of duplex DNA to catalyze the transformation of one adduct into another, which may have important consequences for mutagenesis and repair.