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The 1,4-bis(2'-deoxyadenosin-N(6)-yl)-2S,3S-butanediol intrastrand DNA cross-link arises from the bis-alkylation of tandem N(6)-dA sites in DNA by R,R-butadiene diepoxide (BDO(2)). The oligodeoxynucleotide 5'-d(C(1)G(2)G(3)A(4)C(5)X(6)Y(7)G(8)A(9)A(10)G(11))-3'.5'-d(C(12)T(13)T(14)C(15)T(16)T(17)G(18)T(19)C(20)C(21)G(22))-3' contains the BDO(2) cross-link between the second and third adenines of the codon 61 sequence (underlined) of the human N-ras protooncogene and is named the (S,S)-BD-(61-2,3) cross-link (X,Y = cross-linked adenines). NMR analysis reveals that the cross-link is oriented in the major groove of duplex DNA. Watson-Crick base pairing is perturbed at base pair X(6).T(17), whereas base pairing is intact at base pair Y(7).T(16). The cross-link appears to exist in two conformations, in rapid exchange on the NMR time scale. In the first conformation, the beta-OH is predicted to form a hydrogen bond with T(16) O(4), whereas in the second, the beta-OH is predicted to form a hydrogen bond with T(17) O(4). In contrast to the (R,R)-BD-(61-2,3) cross-link in the same sequence (Merritt, W. K., Nechev, L. V., Scholdberg, T. A., Dean, S. M., Kiehna, S. E., Chang, J. C., Harris, T. M., Harris, C. M., Lloyd, R. S., and Stone, M. P. (2005) Biochemistry 44, 10081-10092), the anti-conformation of the two hydroxyl groups at C(beta) and C(gamma) with respect to the C(beta)-C(gamma) bond results in a decreased twist between base pairs X(6).T(17) and Y(7).T(16), and an approximate 10 degrees bending of the duplex. These conformational differences may account for the differential mutagenicity of the (S,S)- and (R,R)-BD-(61-2,3) cross-links and suggest that stereochemistry plays a role in modulating biological responses to these cross-links (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).

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].

The solution structure of the N1-(1-hydroxy-3-buten-2(S)-yl)-2'-deoxyinosine adduct arising from the alkylation of adenine N1 by butadiene epoxide (BDO), followed by deamination to deoxyinosine, was determined, in the oligodeoxynucleotide d(CGGACXAGAAG).d(CTTCTCGTCCG). This oligodeoxynucleotide contained the BDO adduct at the second position of codon 61 of the human N-ras protooncogene, and was named the ras61 S-N1-BDO-(61,2) adduct. (1)H NMR revealed a weak C(5) H1' to X(6) H8 NOE, followed by an intense X(6) H8 to X(6) H1' NOE. Simultaneously, the X(6) H8 to X(6) H3' NOE was weak. The resonance arising from the T(17) imino proton was not observed. (1)H NOEs between the butadiene moiety and the DNA positioned the adduct in the major groove. Structural refinement based upon a total of 364 NOE-derived distance restraints yielded a structure in which the modified deoxyinosine was in the high syn conformation about the glycosyl bond, and T(17), the complementary nucleotide, was stacked into the helix, but not hydrogen bonded with the adducted inosine. The refined structure provided a plausible hypothesis as to why this N1 deoxyinosine adduct strongly coded for the incorporation of dCTP during trans lesion DNA replication, both in Escherichia coli [Rodriguez, D. A., Kowalczyk, A., Ward, J. B. J., Harris, C. M., Harris, T. M., and Lloyd, R. S. (2001) Environ. Mol. Mutagen. 38, 292-296], and in mammalian cells [Kanuri, M., Nechev, L. N., Tamura, P. J., Harris, C. M., Harris, T. M., and Lloyd, R. S. (2002) Chem. Res. Toxicol. 15, 1572-1580]. Rotation of the N1 deoxyinosine adduct into the high syn conformation may facilitate incorporation of dCTP via Hoogsteen-type templating with deoxyinosine, thus generating A-to-G mutations.

The (2S,3S)-N6-(2,3,4-trihydroxybutyl)-2'-deoxyadenosyl (BDT) adduct arising from alkylation of adenine N6 by butadiene diol epoxide (BDE) was placed opposite a mismatched deoxyguanosine nucleotide in the complementary strand of the oligodeoxynucleotide 5'-d(CGGACXAGAAG)-3'.5'-d(CTTCTGGTCCG)-3'. This oligodeoxynucleotide contains codon 61 (underlined) of the human N-ras protooncogene. The BDT adduct was at the second position of codon 61, and this was named the ras61 S,S-BDT-(61,2) A.G adduct. NMR spectroscopy revealed the presence of two conformations of the adducted mismatched duplex. In the major conformation, the mismatched base pair X6.G17 was oriented in a "face-to-face" orientation, in which both the modified nucleotide X6 and its complement G17 were intrahelical and in the anti conformation about the glycosyl bond. Hydrogen bonding was suggested between X6 N1 and G17 N1H and between X6 N6H and G17 O6. The presence of the BDT moiety allowed formation of a stable A.G mismatch pair. The identity of the minor conformation could not be determined. If not repaired, the resulting mismatch pair would generate A-->C mutations, which have been associated with this adenine N6 BDT adduct [Carmical, J. R., Nechev, L. N., Harris, C. M., Harris, T. M., and Lloyd, R. S. (2000) Env. Mol. Mutagen. 35, 48-56].

Butadiene is oxidized in vivo to form stereoisomeric butadiene diol epoxides (BDE). These react with adenine N(6) in DNA yielding stereoisomeric N(6)-(2,3,4-trihydroxybutyl)-2'-deoxyadenosyl (BDT) adducts. When replicated in Escherichia coli, the (2R,3R)-N(6)-(2,3,4-trihydroxybutyl)-2'-deoxyadenosyl adduct yielded low levels of A-->G mutations whereas the (2S,3S)-N(6)-(2,3,4-trihydroxybutyl)-2'-deoxyadenosyl butadiene triol adduct yielded low levels of A-->C mutations [Carmical, J. R., Nechev, L. V., Harris, C. M., Harris, T. M., and Lloyd, R. S. (2000) Environ. Mol. Mutagen. 35, 48-56]. Accordingly, the structure of the (2R,3R)-N(6)-(2,3,4-trihydroxybutyl)-2'-deoxyadenosyl adduct at position X(6) in d(CGGACXAGAAG).d(CTTCTTGTCCG), the ras61 R,R-BDT-(61,2) adduct, was compared to the corresponding structure for the (2S,3S)-N(6)-(2,3,4-trihydroxybutyl)-2'-deoxyadenosyl adduct in the same sequence, the ras61 S,S-BDT-(61,2) adduct. Both the R,R-BDT-(61,2) and S,S-BDT-(61,2) adducts are oriented in the major groove of the DNA, accompanied by modest structural perturbations. However, structural refinement of the two adducts using a simulated annealing restrained molecular dynamics (rMD) approach suggests stereospecific differences in hydrogen bonding between the hydroxyl groups located at the beta- and gamma-carbons of the BDT moiety, and T(17) O(4) of the modified base pair X(6).T(17). The rMD calculations predict hydrogen bond formation between the gamma-OH and the T(17) O(4) in the R,R-BDT-(61,2) adduct whereas in the S,S-BDT-(61,2) adduct, hydrogen bond formation is predicted between the beta-OH and the T(17) O(4). This difference positions the two adducts differently in the major groove. This may account for the differential mutagenicity of the two adducts and suggests that the two adducts may interact differentially with other DNA processing enzymes. With respect to mutagenesis in E. coli, the minimal perturbation of DNA induced by both major groove adducts correlates with their facile bypass by three E. coli DNA polymerases in vitro and may account for their weak mutagenicity [Carmical, J. R., Nechev, L. V., Harris, C. M., Harris, T. M., and Lloyd, R. S. (2000) Environ. Mol. Mutagen. 35, 48-56].

The solution structure of the (2S,3S)-N(6)-(2,3,4-trihydroxybutyl)-2'-deoxyadenosyl adduct arising from the alkylation of adenine N(6) at position X(6) in d(CGGACXAGAAG).d(CTTCTTGTCCG), by butadiene diol epoxide, was determined. This oligodeoxynucleotide contains codon 61 (underlined) of the human N-ras protooncogene. This oligodeoxynucleotide, containing the adenine N(6) adduct butadiene triol (BDT) adduct at the second position of codon 61, was named the ras61 S,S-BDT-(61,2) adduct. NMR spectroscopy revealed modest structural perturbations localized to the site of adduction at X(6).T(17), and its nearest-neighbor base pairs C(5).G(18) and A(7).T(16). All sequential NOE connectivities arising from DNA protons were observed. Torsion angle analysis from COSY data suggested that the deoxyribose sugar at X(6) remained in the C2'-endo conformation. Molecular dynamics calculations using a simulated annealing protocol restrained by a total of 442 NOE-derived distances and J coupling-derived torsion angles refined structures in which the BDT moiety oriented in the major groove. Relaxation matrix analysis suggested hydrogen bonding between the hydroxyl group located at the beta-carbon of the BDT moiety and the T(17) O(4) of the modified base pair X(6).T(17). The minimal perturbation of DNA induced by this major groove adduct correlated with its facile bypass by three Escherichia coli DNA polymerases in vitro and its weak mutagenicity [Carmical, J. R., Nechev, L. V., Harris, C. M., Harris, T. M., and Lloyd, R. S. (2000) Environ. Mol. Mutagen. 35, 48-56]. Overall, the structure of this adduct is consistent with an emerging pattern in which major groove adenine N(6) alkylation products of styrene and butadiene oxides that do not strongly perturb DNA structure are not strongly mutagenic.