Structure of the 1,4-Bis(2′-deoxyadenosin-N⁶-yl)-2R,3R- butanediol cross-link arising from alkylation of the human N-ras codon 61 by butadiene diepoxide

The solution structure of the 1,4-bis(2′-deoxyadenosin-N ⁶-yl)-2R,3R-butanediol cross-link arising from N⁶-dA alkylation of nearest-neighbor adenines by butadiene diepoxide (BDO₂) was determined in the oligodeoxynucleotide 5′-d(CGGACXYGAAG)-3′- 5′-d(CTTCTTGTCCG)-3′. This oligodeoxynucleotide contained codon 61 (underlined) of the human N-ras protooncogene. The cross-link was accommodated in the major groove of duplex DNA. At the 5′-side of the cross-link there was a break in Watson-Crick base pairing at base pair X⁶· T¹⁷, whereas at the 3′-side of the cross-link at base pair Y⁷·T¹⁶, base pairing was intact. Molecular dynamics calculations carried out using a simulated annealing protocol, and restrained by a combination of 338 interproton distance restraints obtained from ¹H NOESY data and 151 torsion angle restraints obtained from ¹H and ³¹P COSY data, yielded ensembles of structures with good convergence. Helicoidal analysis indicated an increase in base pair opening at base pair X⁶·T¹⁷, accompanied by a shift in the phosphodiester backbone torsion angle β P5′-O5′- C5′-C4′ at nucleotide X⁶. The rMD calculations predicted that the DNA helix was not significantly bent by the presence of the four-carbon cross-link. This was corroborated by gel mobility assays of multimers containing nonhydroxylated four-carbon N⁶,N⁶-dA cross-links, which did not predict DNA bending. The rMD calculations suggested the presence of hydrogen bonding between the hydroxyl group located on the β-carbon of the four-carbon cross-link and T¹⁷ O⁴, which perhaps stabilized the base pair opening at X⁶·T ¹⁷ and protected the T¹⁷ imino proton from solvent exchange. The opening of base pair X⁶·¹⁷ altered base stacking patterns at the cross-link site and induced slight unwinding of the DNA duplex. The structural data are interpreted in terms of biochemical data suggesting that this cross-link is bypassed by a variety of DNA polymerases, yet is significantly mutagenic [Kanuri, M., Nechev, L. V., Tamura, P. J., Harris;, C. M., Harris, T. M., and Lloyd, R. S. (2002) Chem. Res. Toxicol. 15, 1572-1580].