Structure of the 1,4-Bis(2'-deoxyadenosin-N6-yl)-2R,3R-butanediol Crosslink 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)-2 R ,3 R -butanediol crosslink arising from N 6 -dA alkylation of nearest neighbor adenines by butadiene diepoxide (BDO 2 ) was determined in the oligodeoxynucleotide 5' - d(CGGA CXY GAAG)•d(CTTCTCGTCCG)-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 crosslink there was a break in Watson-Crick base pairing at base pair X 6 •T 17 , whereas at the 3'-side of the crosslink at base pair Y 7 •T 16 , 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 1 H NOESY data and 151 torsion angle restraints obtained from 1 H and 31 P COSY data, yielded ensembles of structures with good convergence. Helicoidal analysis indicated an increase in base pair opening at base pair X 6 •T 17 , accompanied by a shift in the phosphodiester backbone torsion angle β P5'-O5'-C5'-C4' at nucleotide X 6 . The rMD calculations predicted that the DNA helix was not significantly bent by the presence of the four-carbon crosslink. This was corroborated by gel mobility assays of multimers containing non-hydroxylated four carbon N 6 ,N 6 -dA crosslinks, 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 fourcarbon crosslink, and T 17 O 4 , which perhaps stabilized the base pair opening at X 6 •T 17 and protected the T 17 imino proton from solvent exchange. The opening of base pair X 6 •T 17 altered base stacking patterns at the crosslink site and induced slight unwinding of the DNA duplex. The structural data are interpreted in terms of biochemical data suggesting that this crosslink 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].