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

W. Keither Merritt, Lubomir V. Nechev, Tandace A. Scholdberg, Stephen M. Dean, Sarah E. Kiehna, Johanna C. Chang, Thomas M. Harris, Constance M. Harris, R. Stephen Lloyd, Michael P. Stone

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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].

Original languageEnglish (US)
Pages (from-to)10081-10092
Number of pages12
Issue number30
StatePublished - Aug 2 2005

ASJC Scopus subject areas

  • Biochemistry


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