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 N(6)-dA alkylation of nearest-neighbor adenines by butadiene diepoxide (BDO(2)) 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 X(6).T(17), whereas at the 3'-side of the cross-link 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 beta 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 cross-link. This was corroborated by gel mobility assays of multimers containing nonhydroxylated four-carbon N(6),N(6)-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 beta-carbon of the four-carbon cross-link 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 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].
Merritt
W Keither
WK
Department of Chemistry, Center in Molecular Toxicology, Vanderbilt-Ingram Cancer Center, Vanderbilt University, Nashville, Tennessee 37235, USA.
Nechev
Lubomir V
LV
Scholdberg
Tandace A
TA
Dean
Stephen M
SM
Kiehna
Sarah E
SE
Chang
Johanna C
JC
Harris
Thomas M
TM
Harris
Constance M
CM
Lloyd
R Stephen
RS
Stone
Michael P
MP
D000477:Alkylating Agents / Q000737:chemistry*; D020029:Base Pairing / Q000187:drug effects; D002070:Butadienes / Q000737:chemistry* / Q000494:pharmacology; D002072:Butylene Glycols / Q000737:chemistry*; D003062:Codon / Q000378:metabolism*; D003432:Cross-Linking Reagents / Q000737:chemistry; D018736:DNA Adducts / Q000737:chemistry*; D003839:Deoxyadenosines / Q000737:chemistry*; D004852:Epoxy Compounds / Q000737:chemistry* / Q000494:pharmacology; D011905:Genes, ras / Q000187:drug effects*; D006801:Humans; D009153:Mutagens / Q000737:chemistry; D019906:Nuclear Magnetic Resonance, Biomolecular; D009692:Nucleic Acid Heteroduplexes / Q000737:chemistry; D009838:Oligodeoxyribonucleotides / Q000737:chemistry; D011522:Protons
C505136:1,4-bis(2'-deoxyadenosin-N6-yl)-2R,3R-butanediol; D000477:Alkylating Agents; D002070:Butadienes; D002072:Butylene Glycols; D003062:Codon; D003432:Cross-Linking Reagents; D018736:DNA Adducts; D003839:Deoxyadenosines; D004852:Epoxy Compounds; D009153:Mutagens; D009692:Nucleic Acid Heteroduplexes; D009838:Oligodeoxyribonucleotides; D011522:Protons; C007366:diepoxybutane; C039681:1,4-butanediol