NMR STRUCTURAL STUDIES OF A 15-MER DNA DUPLEX FROM A RAS PROTOONCOGENE MODIFIED WITH THE CARCINOGEN 2-AMINOFLUORENE - CONFORMATIONAL HETEROGENEITY

Proton NMR studies were conducted on the complementary 15-mer DNA dupl ex, d(5'-TACTCTTCTT[AF]GACCT)-d(5'-AGGTCAAGAAGAGTA) (designated as the AF-modified duplex). The sequence represents a portion of the mouse c -Ha-ras protooncogene and was selectively modified to contain a single N-(deoxyguanosin-8-yl)-2-aminofluorene (dG-C8-AF) adduct at the deoxy guanosine corresponding to the first base of codon 61. The AF-modified duplex was found to exist in multiple conformations, with one being p redominant (similar to 60%). The exchangeable and nonexchangeable prot ons belonging to the major conformer were sufficiently well-resolved t o allow the assignment of the majority of the base and sugar protons. The one-dimensional proton spectra, as well as the NOE cross-peak patt erns associated with this conformer of the AF-modified duplex both in H2O and D2O spectra, were strikingly similar to those observed for the major conformer of an analogous duplex containing N-(deoxyguanosin-8- yl)-4-aminobiphenyl (dG-C8-ABP) in the same position [Cho, B. P., Bela nd, F. A., & Marques, M. M. (1992) Biochemistry 31, 9587-9602]. The ex perimental results suggest that the AF- and ABP-modified duplexes adop t essentially identical major conformations, with each arylamine moiet y being positioned in the major groove of a slightly disturbed B-type DNA duplex. Nonetheless, the absence of specific NOE cross peaks in th e vicinity of the modification site indicates that the local structura l perturbation is more severe in the AF-modified duplex. Although insu fficient data precluded a detailed characterization of the minor confo rmers of the AF-modified duplex, the observation of significant shield ing of the AF aromatic protons suggests a more dramatic structural alt eration at the adduct site, possibly involving extensive stacking with the neighboring bases. The higher content (30-40%) of the minor confo rmers observed for the AF-modified duplex contrasted with the low cont ribution (5-10%) of similar structures in the ABP-modified duplex and may be attributed to a better overlapping efficiency of the planar AF ring with the nearby bases. Since the significant local perturbation o bserved in the minor conformers could provide a possible mechanism for mutations, our results support the view that the structural differenc es in the arylamine fragments of otherwise identical adducts have a di rect influence on the conformational heterogeneities, which in turn ma y play a significant role in arylamine carcinogenesis.