Structural Characterization of Two Interchangeable Conformations of an N-2-Aminofluorene-Modified DNA Oligomer by NMR and Energy Minimization

Abstract

One- and two-dimensional NMR spectroscopy and energy minimization calculations were used to investigate the conformation of a 2-aminofluorene- (AF-) modified model human c-H-ras1 protooncogene codon 61 deoxyoligonucleotide duplex, d(C1-A2-C3-C4-A5- [AF-G6]-G7-A8-A9-C10).-d(G11-T12-T13-C14-C15-T16 -G17-G18-T19-G20), in which the AF adduct is located at the third base of codon 61 with cytosine as the complementary nucleotide. Two interchangeable conformations of the AF-modified duplex, referred to as the external-AF conformation and the inserted-AF conformation, were determined from the NMR data. An analysis of the coalescence of resonances led to the estimation that the chemical exchange lifetime is greater than 3 ms but less than 20 ms at 30 degrees C, pH 7. In the external-AF conformation, Watson-Crick base-pair formation is observed for all 10 complementary nucleotides, including the AF-G6.C15 base pair. In the inserted-AF conformation, 9 of the 10 complementary bases form Watson-Crick base pairs; the AF-G6 imino proton exhibits no evidence of hydrogen bond formation with its complementary cytosine. Several NOEs between aminofluorene protons and DNA protons show that the AF moiety in the inserted-AF conformation stacks between the adjacent A5.T16 and G7.C14 base pairs. Solvated energy minimization calculations using distance restraints obtained from NOESY data at 2 degrees C with a 100-ms mixing time were performed to obtain representative structures of the external-AF and inserted-AF conformations. The external-AF conformer has the AF moiety protruding out of the major groove of a relatively unperturbed DNA duplex, leaving intact Watson-Crick base pairing for the AF-G6.C15 bases. Thus, the external-AF conformer may represent a visualization of a conformation that allows faithful replication. The inserted-AF conformer has the AF moiety stacked within the DNA helix, breaking the Watson-Crick base pairing of the modified guanine and its complementary cytosine and displacing the guanine and cytosine into the grooves. We label the inserted-AF conformer as a premutagenic conformation to reflect the displacement of the modified guanine. Interconversion between the structurally distinct external-AF and inserted-AF conformers takes place on a time scale of the same order as DNA replication. We have labeled this interconversion as a mutagenic switch to highlight a possible conformational equilibrium that may be important in replication.

Solution structure of the 2-amino-1- methyl-6-phenylimidazo[4,5- b ]pyridine C8-deoxyguanosine adduct in duplex DNA

“…Our studies show that the [PhIP]dG⅐dC 11-mer adduct duplex undergoes a conformational exchange between a major base-displaced intercalative structure and a minor external groove-binding structure. Similar distortions have been observed for other DNA adducts, in which the guanine is modified by activated polycyclic aromatic hydrocarbons (30) and aromatic amines (24,34). Here we report that the subtle differences in the structural details and in the population ratios of each conformer of the C8-dG-PhIP-DNA adduct reveal the importance of the chemical structure of the PhIP molecule in governing its DNA adduct conformation.…”

Sequence and Stereospecific Consequences of Major Groove α-(N6-Adenyl)-Styrene Oxide Adducts in an Oligodeoxynucleotide Containing the HumanN-rasCodon 61 Sequence

“…Support for this hypothesis comes from evidence that correct nucleotide insertion during replication is dependent upon attainment of the correct geometry of the incoming nucleotide. 2, 7 That small changes in adduct conformation may correlate with larger biological response reveals the need to discern fine details of adduct structure with a high degree of ~onfidence.~~ The present structures were refined from NOE data (Plate 2) and were generally corroborated by chemical shift perturbations. However, there remained discrepancies, which suggested that the refinement could be improved with the incorporation of additional restraints.…”

Solution Structure of the Aminofluorene [AF]-Intercalated Conformer of the syn-[AF]-C8-dG Adduct Opposite dC in a DNA Duplex

“…They considered both anti and syn alignments for the [AF]-dG residue in the AF-intercalated conformer but eventually favored the anti alignment. The principle reason for this choice was their reasoning that the 10 ms interconversion time scale favored anti alignments for both the AFintercalated and AF-external conformers (14,15). However, using relaxation measurements, chemical-shift considerations, and complete band shape calculations of the exchange process, Ippel and co-workers have revealed the existence of multiconformational states in the anti-syn equilibrium at a similar time scale in DNA hairpin loops (37).…”