Solution Structure of an Oligodeoxynucleotide Containing the Human N-ras Codon 12 Sequence Refined from ¹H NMR Using Molecular Dynamics Restrained by Nuclear Overhauser Effects

Abstract

The structure of d(GGCAGGTGGTG).d(CACCACCTGCC), consisting of codons 11, 12 (underlined), and 13 of the human n-ras protooncogene, was refined from 1H NMR data. Patterns of internucleotide NOEs consistent with a B-form helix were observed for each strand. NOE intensities between purine H8 and H1' protons were small compared to intensities between cytosine H5 and H6 protons, indicative of glycosyl torsion angles in the anti range. Cross-peaks were observed between purine H8 and pyrimidine H5 and CH3 protons on adjacent bases in the direction of purine (5'-->3')pyrimidine, but not in the direction pyrimidine(5'-->3')purine. Watson-Crick hydrogen bonding between bases was intact. A total of 232 experimental distance restraints were obtained. Of these, 143 were intra-residue restraints and 89 were inter-residue restraints. A restrained molecular dynamics/simulated annealing approach yielded 6 MD structures calculated from a B-form starting structure and 6 MD structures from an A-form starting structure. These refined to an average pairwise rms difference of 0.92 angstrom, with maximum pairwise rmsd of 1.35 angstroms. Accuracy of emergent structures was assessed by relaxation matrix back-calculation. The sixth-root residual index of 7.0 x 10(-2) measured between the refined structures and the NOE intensity data suggested that the former were in reasonable agreement with the NOE data. The refined solution structures were in the B-family. Similar to the human n-ras codon 61 sequence [Feng, B., & Stone, M.P. (1995) Chem. Res. Toxicol. 8, 821-832], the ras12 sequence contained local variations in B-like conformation which did not confer large structural alterations upon the duplex, but perhaps modulated the reactivity of the first as compared to the second guanine in codon 12.

Thermodynamic and structural factors in the removal of bulky DNA adducts by the nucleotide excision repair machinery

“…20,21 The 10R(Ϫ)-trans-anti adduct structure shown is essentially the same as that of the NMR solution structure of Zegar et al 22 determined in the same C[A*]A sequence context of codon 61 of the human N-ras gene.…”

Styrene Oxide Adducts in an Oligodeoxynucleotide Containing the Human N-ras Codon 12: Minor Groove Structures of the R(12,1)- and S(12,1)-α-(N2-Guanyl) Stereoisomers Determined by 1H Nuclear Magnetic Resonance

“…The 3′-neighbor G‚C base pair inserts the guanine exocyclic NH 2 group into the minor groove; thus, accommodation of the styrenyl moiety within the minor groove and in the 3′-direction requires a shift in base stacking at the adduct site. Modeling suggests that the modified base pair R-SO G‚C shifts toward the minor groove as compared to the corresponding G‚C base pair in the unmodified ras12 oligodeoxynucleotide (24). In contrast, with the R(12,2) adduct, the 3′-neighbor T‚A base pair does not hinder accommodation of the styrenyl moiety within the minor groove and in the 3′-direction.…”

“…The greatest changes in chemical shifts were observed for G 6 (Figure 5), in the 3′-direction from the adduct site. Upfield chemical shifts of 0.3, 0.6, and 0.5 ppm were observed for G 6 H8, H1′, and H2′′, respectively, relative to the unmodified oligomer (24). Also, a 0.4 ppm downfield chemical shift was noted for R-SO G 5 H1′.…”

“…We have focused attention on the R-and S-R-SO adducts located at the exocyclic amino groups of guanines in the codon 12 sequence of the human N-ras protooncogene, a site at which mutations are linked to oncogene activation. The structures of the ras12 oligodeoxynucleotide d(GGCAGGTGGTG)‚d(CACCACCTGCC) 2 (24) and the R-and S- (12,2)-R-styrene oxide lesions in the ras12 oligodeoxynucleotide (25) revealed the influence of stereochemistry in determining the orientation of R-SO adducts at guanine N 2 , with the R-diastereomer being oriented in the 3′-direction while the S-diastereomer was oriented in the 5′-direction from the lesion site. That work suggested the potential for flanking base effects upon the R-SO and S-SO adduct minor groove adducts in the ras12 sequence.…”

Repair-Resistant DNA Lesions

“…The NMR solution structures of these diastereomeric B­[ a ]­PDE- N 6 -adenine adducts (dA*) in double-stranded oligonucleotides have been evaluated in the CA*C ,,, and CAA* ,,,, sequence contexts and are very different from those of the N 2 -guanine adducts. In the case of the 10 R (−)- trans -B­[ a ]­PDE- N 6 –dA adduct, all Watson–Crick base pairs, including the modified dA*:dT base pair, are intact. , The dA* residue is predominantly (95%) in the anti glycosidic bond conformation that is in equilibrium with a minor syn conformation.…”