Sequence- and stereospecific conformational rearrangement of styrene oxide adducts located at A x C mismatched base pairs

The solution structures of R- and S-alpha-(N(6)-adenyl)-styrene oxide adducts mismatched with cytosine at position X(7) in d(CGGACAXGAAG) x d(CTTCCTGTCCG), incorporating codons 60, 61 (underlined), and 62 of the human N-ras protooncogene, were determined. These were the R- and S(61,3)C adducts. The structures for these mismatched adducts differed from the sequence isomeric R- and S(61,2)C adducts [Painter, S. L., Zegar, I. S., Tamura, P. J., Bluhm, S., Harris, C. M., Harris, T. M., and Stone, M. P. (1999) Biochemistry 38, 8635-8646]. The results reveal that the structural consequences of cytosine mispairing opposite the R- and S-alpha-SO adducts differ as a function of DNA sequence. The thermodynamic stability of both the R- and S(61,3)C mismatched adducts was dependent upon pH. At neutral pH, the R- and S(61,3)C adducts exhibited significant structural perturbation and had lower T(m) values, as compared to the R- and S(61,2)C adducts. In both instances, this was attributed to reorientation about the C6-N(6) bond, such that the N(6)H proton faced away from the Watson-Crick face of the purine base and into the major groove. The conformation about the N(6)-C(alpha)-C(beta)-O torsion angle was predicted from rMD calculations to be stabilized by a N/O gauche-type interaction between the styrenyl hydroxyl moiety and adenine N(6) at the lesion site. For the R(61,3)C adduct, the styrenyl moiety remained oriented in the major groove and faced in the 3'-direction. In the properly base-paired R(61,3) adduct, it had faced in the 5' direction. For the S(61,3)C adduct, the styrene ring was inserted into the duplex, approximately perpendicular to the helical axis of the DNA. It faced in the 5'-direction. In the properly base-paired S(61,3) adduct, it had faced in the 3'-direction. The results were correlated with site-specific mutagenesis experiments in vivo. The latter revealed that the R- and S(61,3)-alpha-styrene oxide adducts were nonmutagenic. This may be a consequence of the greater structural perturbation associated with formation of the cytosine mismatch at neutral pH for the R- and S(61,3) adducts as compared to the S(61,2) adduct that exhibited low levels of A --> G mutations.     

Structure of an oligodeoxynucleotide containing a butadiene oxide-derived N1 beta-hydroxyalkyl deoxyinosine adduct in the human N-ras codon 61 sequence

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.     

Wobble dC.dA pairing 5' to the cationic guanine N7 8,9-dihydro-8-(N7-guanyl)-9-hydroxyaflatoxin B1 adduct: implications for nontargeted AFB1 mutagenesis

The structure of 5'-d(ACATC(AFB)GATCT)-3'.5'-d(AGATCAATGT)-3', containing the C(5).A(16) mismatch at the base pair 5' to the modified (AFB)G(6), was determined by NMR. The characteristic 5'-intercalation of the AFB(1) moiety was maintained. The mismatched C(5).A(16) pair existed in the wobble conformation, with the C(5) imino nitrogen hydrogen bonded to the A(16) exocyclic amino group. The wobble pair existed as a mixture of protonated and nonprotonated species. The pK(a) for protonation at the A(16) imino nitrogen was similar to that of the C(5).A(16) wobble pair in the corresponding duplex not adducted with AFB(1). Overall, the presence of AFB(1) did not interfere with wobble pair formation at the mismatched site. Molecular dynamics calculations restrained by distances derived from NOE data and torsion angles derived from (1)H (3)J couplings were carried out for both the protonated and nonprotonated wobble pairs at C(5).A(16). Both sets of calculations predicted the A(16) amino group was within 3 A of the C(5) imino nitrogen. The calculations suggested that protonation of the C(5).A(16) wobble pair should shift C(5) toward the major groove and shift A(16) toward the minor groove. The NMR data showed evidence for the presence of a minor conformation characterized by unusual NOEs between T(4) and (AFB)G(6). T(4) is two nucleotides in the 5'-direction from the modified base. These NOEs suggested that in the minor conformation nucleotide T(4) was in closer proximity to (AFB)G(6) than would be expected for duplex DNA. Modeling studies examined the possibility that T(4) transiently paired with the mismatched A(16), allowing it to come within NOE distance of (AFB)G(6). This model structure was consistent with the unusual NOEs associated with the minor conformation. The structural studies are discussed in relationship to nontargeted C --> T transitions observed 5' to the modified (AFB)G in site-specific mutagenesis experiments [Bailey, E. A., Iyer, R. S., Stone, M. P., Harris, T. M., and Essigmann, J. M. (1996) Proc. Natl. Acad. Sci. U.S.A. 93, 1535-1539].     

Intercalation of the (1R,2S,3R,4S)-N6-[1-(1,2,3,4-tetrahydro-2,3,4-trihydroxybenz[a]anthracenyl)]-2'-deoxyadenosyl adduct in the N-ras codon 61 sequence: DNA sequence effects

The structure of the bay region (1R,2S,3R,4S)-N6-[1-(1,2,3,4-tetrahydro-2,3,4-trihydroxybenz[a]anthracenyl)]-2'-deoxyadenosyl adduct at X(7) of 5'-d(CGGACAXGAAG)-3'.5'-d(CTTCTTGTCCG)-3', incorporating codons 60, 61 (underlined), and 62 of the human N-ras protooncogene, was determined by NMR. This was the bay region benz[a]anthracene RSRS (61,3) adduct. The BA moiety intercalated above the 5'-face of the modified base pair. NOE connectivities between imino protons were disrupted at T16 and T17. Large chemical shifts at the lesion site were consistent with ring current shielding arising from the BA moiety. A large chemical shift dispersion was observed for the BA aromatic protons. An increased rise of 8.17 A was observed between base pairs A6 x T17 and X7 x T(16). The PAH moiety stacked with the purine ring of A6, the 5'-neighbor nucleotide. This resulted in buckling of the 5'-neighbor A6 x T17 base pair, evidenced by exchange broadening for the T17 imino resonance. It also interrupted sequential NOE connectivities between nucleotides C5 and A6. The A6 deoxyribose ring showed an increased percentage of the C3'-endo conformation. This differed from the bay region BA RSRS (61,2) adduct, in which the lesion was located at position X6 [Li, Z., Mao, H., Kim, H.-Y., Tamura, P. J., Harris, C. M., Harris, T. M., and Stone, M. P. (1999) Biochemistry 38, 2969-2981], but was similar to the benzo[a]pyrene BP SRSR (61,3) adduct [Zegar I. S., Chary, P., Jabil, R. J., Tamura, P. J., Johansen, T. N., Lloyd, R. S., Harris, C. M., Harris, T. M., and Stone, M. P. (1998) Biochemistry 37, 16516-16528]. The altered sugar pseudorotation at A6 appears to be common to both bay region BA RSRS (61,3) and BP SRSR (61,3) adducts. It could not be discerned if the C3'-endo conformation at A6 in the BA RSRS (61,3) adduct altered base pairing geometry at X7 x T16, as compared to the C2'-endo conformation. The structural studies suggest that the mutational spectrum of this adduct may be more complex than that of the BA RSRS (61,2) adduct.     

Intercalation of the (-)-(1R,2S,3R, 4S)-N6-[1-benz[a]anthracenyl]-2'-deoxyadenosyl adduct in an oligodeoxynucleotide containing the human N-ras codon 61 sequence

The solution structure of the (-)-(1R,2S,3R,4S)-N6-[1-(1,2,3, 4-tetrahydroxy-benz[a]anthracenyl)]-2'-deoxyadenosyl adduct at X6 of 5'-d(CGGACXAGAAG)-3'.5'-d(CTTCTTGTCCG)-3', incorporating codons 60, 61(italic), and 62 of the human N-ras protooncogene, was determined. This adduct results from the trans opening of 1S,2R,3R,4S-1, 2-epoxy-1,2,3,4-tetrahydro-benz[a]anthracenyl-3,4-diol by the exocyclic N6 of adenine. Molecular dynamics simulations were restrained by 509 NOEs from 1H NMR. The precision of the refined structures was monitored by pairwise root-mean-square deviations which were <1.2 A; accuracy was measured by complete relaxation matrix calculations, which yielded a sixth root R factor of 9.1 x 10(-)2 at 250 ms. The refined structure was a right-handed duplex, in which the benz[a]anthracene moiety intercalated from the major groove between C5.G18 and R,S,R,SA6.T17. In this orientation, the saturated ring of BA was oriented in the major groove of the duplex, with the aromatic rings inserted into the duplex such that the terminal ring of BA threaded the duplex and faced toward the minor groove direction. The duplex suffered localized distortion at and immediately adjacent to the adduct site, evidenced by the increased rise of 8.8 A as compared to the value of 3.5 A normally observed for B-DNA between base pairs C5.G18 and R,S,R,SA6.T17. These two base pairs also buckled in opposite directions away from the intercalated BA moiety. The refined structure was similar to the (-)-(7S,8R,9S,10R)-N6-[10-(7,8,9, 10)-tetrahydrobenzo[a]pyrenyl)]-2'-deoxyadenosyl adduct of corresponding stereochemistry at X6 of the same oligodeoxynucleotide [Zegar, I. S., Kim, S. J., Johansen, T. N., Horton, P. J., Harris, C. M., Harris, T. M., and Stone, M. P. (1996) Biochemistry 35, 6212-6224]. Both adducts intercalated toward the 5'-direction from the site of adduction. The similarities in solution structures were reflected in similar biological responses, when repair-deficient AB2480 Escherichia coli were transformed with M13mp7L2 DNA site-specifically modified with these two adducts.     

The nonmutagenic (R)- and (S)-beta-(N(6)-adenyl)styrene oxide adducts are oriented in the major groove and show little perturbation to DNA structure

Conformations of (R)-beta-(N(6)-adenyl)styrene oxide and (S)-beta-(N(6)-adenyl)styrene oxide adducts at position X(6) in d(CGGACXAGAAG).d(CTTCTTGTCCG), incorporating codons 60, 61 (underlined), and 62 of the human N-ras protooncogene, were refined from (1)H NMR data. These were designated as the beta-R(61,2) and beta-S(61,2) adducts. A total of 533 distance restraints and 162 dihedral restraints were used for the molecular dynamics calculations of the beta-S(61,2) adduct, while 518 distances and 163 dihedrals were used for the beta-R(61,2) adduct. The increased tether length of the beta-adducts results in two significant changes in adduct structure as compared to the corresponding alpha-styrenyl adducts [Stone, M. P., and Feng, B. (1996) Magn. Reson. Chem. 34, S105-S114]. First, it reduces the distortion introduced into the DNA duplex. For both the beta-R(61,2) and beta-S(61,2) adducts, the styrenyl moiety was positioned in the major groove of the duplex with little steric hindrance. Second, it mutes the influence of stereochemistry at the alpha-carbon such that both the beta-R(61,2) and beta-S(61,2) adducts exhibit similar conformations. The results were correlated with site-specific mutagenesis experiments that revealed the beta-R(61,2) and beta-S(61,2) adducts were not mutagenic and did not block polymerase bypass.     

Role of a polycyclic aromatic hydrocarbon bay region ring in modulating DNA adduct structure: the non-bay region (8S,9R,10S, 11R)-N(6)-[11-(8,9,10,11-tetrahydro-8,9, 10-trihydroxybenz[a]anthracenyl)]-2'-deoxyadenosyl adduct in codon 61 of the human N-ras protooncogene.

The structure of the non-bay region (8S,9R,10S,11R)-N(6)-[11-(8,9,10, 11-tetrahydro-8,9,10-trihydroxybenz[a]anthracenyl)]-2'-de oxyadenosyl adduct at X(6) of 5'-d(CGGACXAGAAG)-3'.5'-d(CTTCTTGTCCG)-3', incorporating codons 60, 61 (underlined), and 62 of the human N-ras protooncogene, was determined. Molecular dynamics simulations were restrained by 475 NOEs from (1)H NMR. The benz[a]anthracene moiety intercalated above the 5'-face of the modified base pair and from the major groove. The duplex suffered distortion at and immediately adjacent to the adduct site. This was evidenced by the disruption of the Watson-Crick base pairing for X(6) x T(17) and A(7) x T(16) and the increased rise of 7.7 A between base pairs C(5) x G(18) and X(6) x T(17). Increased disorder was observed as excess line width of proton resonances near the lesion site. Comparison with the bay region benzo[a]pyrene [Zegar, I. S., Kim, S. J., Johansen, T. N., Horton, P. J., Harris, C. M., Harris, T. M., and Stone, M. P. (1996) Biochemistry 35, 6212-6224] and bay region benz[a]anthracene [Li, Z., Mao, H., Kim, H.-Y., Tamura, P. J., Harris, C. M., Harris, T. M., and Stone, M. P. (1999) Biochemistry 38, 2969-2981] adducts with the corresponding stereochemistry and at the same site shows that this non-bay region benz[a]anthracene lesion assumes different base pair geometry, in addition to exhibiting greater disorder. These differences are attributed to the loss of the bay region ring. The results suggest the bay region ring contributes to base stacking interactions at the lesion site. These structural differences between the non-bay and bay region lesions are correlated with site-specific mutagenesis data. The bay region benzo[a]pyrene and bay region benz[a]anthracene adducts were poorly replicated in vivo, and induced A --> G mutations. In contrast, the non-bay region benz[a]anthracene adduct was easily bypassed in vivo and was nonmutagenic.     

Intercalation of the (1S,2R,3S,4R)-N6-[1-(1,2,3,4-tetrahydro-2,3, 4-trihydroxybenz[a]anthracenyl)]-2'-deoxyadenosyl adduct in an oligodeoxynucleotide containing the human N-ras codon 61 sequence

The (1S,2R,3S,4R)-N(6)-[1-(1,2,3,4-tetrahydro-2,3, 4-trihydroxybenz[a]anthracenyl)]-2'-deoxyadenosyl adduct at X6 of 5'-d(CGGACXAGAAG)-3'.5'-d(CTTCTTGTCCG)-3', incorporating codons 60, 61 (underlined), and 62 of the human N-ras protooncogene, results from trans opening of (1R,2S,3S,4R)-1,2-epoxy-1,2,3, 4-tetrahydrobenz[a]anthracenyl-3,4-diol by the exocyclic N6 of adenine. Two conformations of this adduct exist, in slow exchange on the NMR time scale. A structure for the major conformation, which represents approximately 80% of the population, is presented. In this conformation, an anti glycosidic torsion angle is observed for all nucleotides, including S,R,S,RA6. The refined structure is a right-handed duplex, with the benz[a]anthracene moiety intercalated on the 3'-face of the modified base pair, from the major groove. It is located between S,R,S,RA6.T17 and A7.T16. Intercalation is on the opposite face of the modified S,R,S,RA6.T17 base pair as compared to the (1R,2S,3R,4S)-N6-[1-(1,2,3,4-tetrahydro-2, 3,4-trihydroxybenz[a]anthracenyl)]-2'-deoxyadenosyl adduct, which intercalated 5' to the modified R,S,R,SA6.T17 base pair [Li, Z. , Mao, H., Kim, H.-Y., Tamura, P. J., Harris, C. M., Harris, T. M., and Stone, M. P. (1999) Biochemistry 38, 2969-2981]. The spectroscopic data do not allow refinement of the minor conformation, but suggest that the adenyl moiety in the modified nucleoti111S,R, S,RA6 adopts a syn glycosidic torsion angle. Thus, the minor conformation may create greater distortion of the DNA duplex. The results are discussed in the context of site-specific mutagenesis studies which reveal that the S,R,S,RA6 lesion is less mutagenic than the R,S,R,SA6 lesion.     

Mispairing of the 8,9-dihydro-8-(N7-guanyl)-9-hydroxy-aflatoxin B1 adduct with deoxyadenosine results in extrusion of the mismatched dA toward the major groove

The G --> T transversion is the dominant mutation induced by the cationic trans-8,9-dihydro-8-(N7-guanyl)-9-hydroxy-aflatoxin B(1) adduct. The structure of d(ACATC(AFB)GATCT).d(AGATAGATGT), in which the cationic adduct was mismatched with deoxyadenosine, was refined using molecular dynamics calculations restrained by NOE data and dihedral restraints obtained from NMR spectroscopy. Restrained molecular dynamics calculations refined structures with pairwise rmsd <1 A and a sixth root R1x factor between the refined structure and NOE data of 10.5 x 10-2. The mismatched duplex existed in a single conformation at neutral pH. The aflatoxin moiety intercalated above the 5' face of the modified (AFB)G. The mismatched dA was in the anti conformation about the glycosyl bond. It extruded toward the major groove and did not participate in hydrogen bonding with (AFB)G. The structure was compared with that of d(ACATCGATCT).d(AGATAGATGT) containing the corresponding unmodified G.A mismatch and with d(ACATC(AFB)GATCT).d(AGATCGATGT) containing the aflatoxin lesion in the correctly paired (AFB)G.C context. The correctly paired oligodeoxynucleotide exhibited Watson-Crick-type geometry at the (AFB)G.C pair. It melted at higher temperature than the mismatched (AFB)G.A duplex. The unmodified mismatched G.A duplex exhibited spectral line broadening at neutral pH, suggesting a mixture of conformations. It exhibited a lower melting temperature than did the mismatched (AFB)G.A duplex. These differences correlated with replication bypass experiments performed in vitro utilizing DNA polymerase I exo- [Johnston, D. S., and Stone, M. P. (2000) Chem. Res. Toxicol. 13, 1158-1164]. Those experiments showed that correct insertion of dC opposite (AFB)G blocked replication by the enzyme, whereas incorrect insertion of dA opposite (AFB)G allowed full-length replication of the adducted template strand.     

The exocyclic 1,N2-deoxyguanosine pyrimidopurinone M1G is a chemically stable DNA adduct when placed opposite a two-base deletion in the (CpG)3 frameshift hotspot of the Salmonella typhimurium hisD3052 gene.

The pyrimidopurinone adduct M1G [3-(2'-deoxy-beta-D-erythro-pentofuranosyl)pyrimido[1,2-a]-purin-10(3H)-one], formed in DNA upon exposure to malondialdehyde or base propenals, was incorporated into 5'-d(ATCGCMCGGCATG)-3'-5'-d(CATGCCGCGAT)-3', where M = M1G. This duplex contained a two-nucleotide bulge in the modified strand, and was named the M1G-2BD oligodeoxynucleotide. It provided a model for -2 bp strand slippage deletions associated with the (CpG)3-iterated repeat hotspot for frameshift mutations from the Salmonella typhimurium hisD3052 gene. M1G was chemically stable in the M1G-2BD duplex at neutral pH. The two-base bulge in the M1G-2BD oligodeoxynucleotide was localized and consisted of M1G and the 3'-neighbor deoxycytosine. The intrahelical orientation of M1G was established from a combination of NOE and chemical shift data. M1G was in the anti conformation about the glycosyl bond. The 3'-neighbor deoxycytosine appeared to be extruded toward the major groove. In contrast, when M1G was placed into the corresponding fully complementary (CpG)3-iterated repeat duplex at neutral pH, spontaneous and quantitative ring-opening to N(2)-(3-oxo-1-propenyl)-dG (the OPG adduct) was facilitated [Mao, H., Reddy, G. R., Marnett, L. J., and Stone, M. P. (1999) Biochemistry 38, 13491-13501]. The structure of the M1G-2BD duplex suggested that the bulged sequence lacked a cytosine amino group properly positioned to facilitate opening of M1G and supports the notion that proper positioning of deoxycytosine complementary to M1G is necessary to promote ring-opening of the exocyclic adduct in duplex DNA. The structure of the M1G-2BD duplex was similar to that of the structural analogue 1,N(2)-propanodeoxyguanosine (PdG) in the corresponding PdG-2BD duplex [Weisenseel, J. P., Moe, J. G., Reddy, G. R., Marnett, L. J., and Stone, M. P. (1995) Biochemistry 34, 50-64]. The fixed position of the bulged bases in both instances suggests that these exocyclic adducts do not facilitate transient bulge migration.     

Minor groove orientation for the (1S,2R,3S,4R)-N2- [1-(1,2,3,4-tetrahydro-2,3,4-trihydroxybenz[a]anthracenyl)]-2'-deoxyguanosyl adduct in the N-ras codon 12 sequence

The conformation of the trans-anti-(1S,2R,3S,4R)-N(2)-[1-(1,2,3,4-tetrahydro-2,3,4-trihydroxybenz[a]anthracenyl)]-2'-deoxyguanosyl adduct in d(G(1)G(2)C(3)A(4)G(5)X(6)T(7)G(8)G(9)T(10)G(11)).d(C(12)A(13)C(14)C(15)A(16)C(17)C(18)T(19)G(20)C(21)C(22)), bearing codon 12 of the human N-ras protooncogene (underlined), was determined. This adduct had S stereochemistry at the benzylic carbon. Its occurrence in DNA is a consequence of trans opening by the deoxyguanosine amino group of (1R,2S,3S,4R)-1,2-epoxy-1,2,3,4-tetrahydrobenz[a]anthracenyl-3,4-diol. The resonance frequencies, relative to the unmodified DNA, of the X(6) H1' and H6 protons were shifted downfield, whereas those of the C(18) and T(19) H1', H2', H2' ', and H3' deoxyribose protons were shifted upfield. The imino and amino resonances exhibited the expected sequential connectivities, suggesting no interruption of Watson-Crick pairing. A total of 426 interproton distances, including nine uniquely assigned BA-DNA distances, were used in the restrained molecular dynamics calculations. The refined structure showed that the benz[a]anthracene moiety bound in the minor groove, in the 5'-direction from the modified site. This was similar to the (+)-trans-anti-benzo[a]pyrene-N(2)-dG adduct having S stereochemistry at the benzylic carbon [Cosman, M., De Los Santos, C., Fiala, R., Hingerty, B. E., Singh, S. B., Ibanez, V., Margulis, L. A., Live, D., Geacintov, N. E., Broyde, S., and Patel, D. J. (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 1914-1918]. It differed from the (-)-trans-anti-benzo[c]phenanthrene-N(2)-dG adduct having S stereochemistry at the benzylic carbon, which intercalated in the 5'-direction [Lin, C. H., Huang, X., Kolbanovskii, A., Hingerty, B. E., Amin, S., Broyde, S., Geacintov, N. E., and Patel, D. J. (2001) J. Mol. Biol. 306, 1059-1080]. The results provided insight into how PAH molecular topology modulates adduct structure in duplex DNA.     

Intercalation of aflatoxin B1 in two oligodeoxynucleotide adducts: comparative 1H NMR analysis of d(ATCAFBGAT).d(ATCGAT) and d(ATAFBGCAT)2

8,9-Dihydro-8-(N7-guanyl-[d(ATCGAT)])-9-hydroxyaflatoxin B1.d(ATCGAT) and 8,9-dihydro-8-(N7-guanyl-[d(ATGCAT)])-9-hydroxyaflatoxin B1.8,9-dihydro-8-(N7-guanyl-[d(ATGCAT)])-9-hydroxyaflatoxin B1 were prepared by direct addition of afltoxin B1 8,9-epoxide to d(ATCGAT)2 and d(ATGCAT)2, respectively. In contrast to reaction of aflatoxin B1 8,9-epoxide with d(ATCGAT)2 which exhibits a limiting stoichiometry of 1:1 aflatoxin B1:d(ATCGAT)2 [Gopalakrishnan, S., Stone, M. P., & Harris, T. M. (1989) J. Am. Chem. Soc. 111, 7232-7239], reaction of aflatoxin B1 8,9-epoxide with d(ATGCAT)2 exhibits a limiting stoichiometry of 2:1 aflatoxin B1:d(ATGCAT)2. 1H NOE experiments, nonselective 1H T1 relaxation measurements, and 1H chemical shift perturbations demonstrate that in both modified oligodeoxynucleotides the aflatoxin moiety is intercalated above the 5'-face of the modified guanine. The oligodeoxynucleotides remain right-handed, and perturbation of the B-DNA structure is localized adjacent to the adducted guanine. Aflatoxin-oligodeoxynucleotide 1H NOEs are observed between aflatoxin and the 5'-neighbor base pair and include both the major groove and the minor groove. The aflatoxin methoxy and cyclopentenone ring protons face into the minor groove; the furofuran ring protons face into the major groove. No NOE is observed between the imino proton of the modified base pair and the imino proton of the 5'-neighbor base pair; sequential NOEs between nucleotide base and deoxyribose protons are interrupted in both oligodeoxynucleotide strands on the 5'-side of the modified guanine. The protons at C8 and C9 of the aflatoxin terminal furan ring exhibit slower spin-lattice relaxation as compared to other oligodeoxynucleotide protons, which supports the conclusion that they face into the major groove. Increased shielding is observed for aflatoxin protons; chemical shift perturbations of the oligodeoxynucleotide protons are confined to the immediate vicinity of the adducted base pair. The imidazole proton of the modified guanine exchanges with water and is observed at 9.75 ppm. The difference in reaction stoichiometry is consistent with an intercalated transition-state complex between aflatoxin B1 8,9-epoxide and B-DNA. Insertion of aflatoxin B1-8,9 epoxide above the 5'-face of guanine in d(ATCGAT)2 would prevent the binding of a second molecule of aflatoxin B1 8,9-epoxide. In contrast, two intercalation sites would be available with d(ATGCAT)2.(ABSTRACT TRUNCATED AT 400 WORDS)     

Structure of the anthramycin-d(ATGCAT)2 adduct from one- and two-dimensional proton NMR experiments in solution

One- and two-dimensional 400-MHz proton NMR experiments are used to examine the solution structure of the covalent adduct formed by the interaction of anthramycin methyl ether with the self-complementary deoxyoligonucleotide d(ATGCAT)2. The concentration dependence of chemical shifts and nuclear Overhauser enhancement (NOE) experiments are utilized to assign the adenine H2 protons within the minor groove for both free d(ATGCAT)2 and the adduct. These studies demonstrate that one of the four adenine H2 protons is in close proximity to the bound anthramycin and this results in its upfield shift of 0.3 ppm compared to the adenine H2 protons of the free duplex. Effects of the covalent attachment of anthramycin to the d(ATGCAT)2 duplex result in an increased shielding of selected deoxyribose protons located within the minor groove of the adduct, as demonstrated by two-dimensional autocorrelated (COSY) NMR techniques. Interactions between the protons of the covalently attached anthramycin and the d(ATGCAT)2 duplex are determined by utilizing two-dimensional NOE (NOESY) techniques. Analysis of these data reveals NOE cross-peaks between the anthramycin methyl, H6, and H7 protons with specific deoxyoligonucleotide protons within the minor groove, thus allowing the orientation of the drug within the minor groove to be determined. Nonselective inversion recovery (T1) relaxation experiments are used to probe the structural and dynamic properties of the anthramycin-d(ATGCAT)2 adduct. These data suggest that the binding of anthramycin alters the correlation time of the d(ATGCAT)2 duplex and stabilizes both of the internal A X T base pairs with respect to solvent exchange.(ABSTRACT TRUNCATED AT 250 WORDS)     

1H two-dimensional nuclear Overhauser effect and relaxation studies of poly(dA).poly(dT)

The structure of poly(dA).poly(dT) in aqueous solution has been studied by using 1H two-dimensional nuclear Overhauser effect (2D NOE) spectroscopy and relaxation rate measurements on the imino and nonexchangeable protons. The assignments of the 1H resonances are determined from the observed cross-relaxation patterns in the 2D NOE experiments. The cross-peak intensities together with the measured relaxation rates show that the purine and pyrimidine strands in poly(dA).poly(dT) are equivalent in aqueous solution. The results are consistent with a right-handed B-form helix where the sugars on both strands are in the C2'-endo/anti configuration. These observations are inconsistent with a proposed heteronomous structure for poly(dA).poly(dT) [Arnott, S., Chandrasekaran, R., Hall, I. H., & Puigjaner, L. C. (1983) Nucleic Acids Res. 11, 4141-4155]. The measured relaxation rates also show that poly(dA).poly(dT) has fast, large-amplitude local internal motions (+/- 20-25 degrees) in solution and that the amplitudes of the base and sugar motions are similar. The motion of the bases in poly(dA).poly(dT) is also similar to that previously reported for poly(dA-dT).poly(dA-dT) and poly(dG-dC).poly(dG-dC) [Assa-Munt, N., Granot, J., Behling, R. W., & Kearns, D. R. (1984) Biochemistry 23, 944-955; Mirau, P. A., Behling, R. W., & Kearns, D. R. (1985) Biochemistry 24, 6200-6211].     

The structure of d(TpA), the major photoproduct of thymidylyl-(3'5')-deoxyadenosine.

Irradiation of the dinucleotide TpdA and TA-containing oligonucleotides and DNA produces the TA* photoproduct which was proposed to be the [2+2] cyclo-addition adduct between the C5-C6 double bonds of the T and the A [Bose,S.N., Kumar,S., Davies,R.J.H., Sethi,S.K. and McCloskey,J.A. (1984) Nucleic Acids Res. 12, 7929-7947]. The proposed structure was based on a variety of spectroscopic and chemical degradation studies, and the assignment of a trans-syn-I stereochemistry was based on an extensive 1H-NMR and molecular modeling study of the dinucleotide adduct [Koning,T.M.G., Davies,R.J.H. and Kaptein,R. (1990) Nucleic Acids Res. 18, 277-284]. However, a number of properties of TA* are not in accord with the originally proposed structure, and prompted a re-evaluation of the structure. To assign the 13C spectrum and establish the bond connectivities of the TA* photoproduct of TpdA [d(TpA)*], 1H-13C heteronuclear multiple-quantum coherence (HMQC) and heteronuclear multiple bond correlation (HMBC) spectra were obtained. The 13C shifts and connectivities were found to be inconsistent with the originally proposed cyclobutane ring fusion between the thymine and adenine, but could be explained by a subsequent ring-expansion reaction to give an eight-membered ring valence isomer. The new structure for the d(TpA)* resolves the inconsistencies with the originally proposed structure, and could have a stereochemistry that arises from the anti, anti glycosyl conformation found in B form DNA.     

Solution structure of the covalent sterigmatocystin-DNA adduct.

Sterigmatocystin and aflatoxin are potent mutagens that contaminate foodstuffs stored under conditions that permit fungal growth. These food mycotoxins can be metabolically activated to their epoxides, which subsequently form covalent adducts with DNA and can eventually induce tumor development. We have generated the sterigmatocystin-d(A1-A2-T3-G4-C5-A6-T7-T8) covalent adduct (two sterigmatocystins per duplex) by reacting sterigmatocystin-1,2-epoxide with the self-complementary d(A-A-T-G-C-A-T-T) duplex and determined its solution structure by the combined application of two-dimensional NMR experiments and molecular dynamics calculations. The self-complementary duplex retains its 2-fold symmetry following covalent adduct formation of sterigmatocystin at the N7 position of G4 residues on each strand of the duplex. The H8 proton of [ST]G4 exchanges rapidly with water and resonates at 9.58 ppm due to the presence of the positive charge on the guanine ring following adduct formation. We have assigned the exchangeable and nonexchangeable proton resonances of sterigmatocystin and the duplex in the covalent adduct and identified the intermolecular proton-proton NOEs that define the orientation and mode of binding of the mutagen to duplex DNA. The analysis was aided by intermolecular NOEs between the sterigmatocystin protons with both the major groove and minor groove protons of the DNA. The molecular dynamics calculations were aided by 180 intramolecular nucleic acid constraints, 16 intramolecular sterigmatocystin constraints, and 56 intermolecular distance constraints between sterigmatocystin and the nucleic acid protons in the adduct. The sterigmatocystin chromophore intercalates between the [ST]G4.C5 and T3.A6 base pairs and stacks predominantly over the modified guanine ring in the adduct duplex. The overall conformation of the DNA remains right-handed on adduct formation with unwinding of the helix, as well as widening of the minor groove. Parallel NMR studies on the sterigmatocystin-d(A1-A2-A3-G4-C5-T6-T7-T8) covalent adduct (two sterigmatocystins per duplex) provide supportive evidence that the mutagen covalently adducts the N7 position of G4 and its chromophore intercalates to the 5' side of the guanine and stacks over it. The present NMR-molecular dynamics studies that define a detailed structure for the sterigmatocystin-DNA adduct support key structural conclusions proposed previously on the basis of a qualitative analysis of NMR parameters for the adduct formed by the related food mutagen aflatoxin B1 and DNA [Gopalakrishnan, S., Harris, T. M., & Stone, M. P. (1990) Biochemistry 29, 10438-10448].     

Solution structure of a DNA duplex containing a cis-diammineplatinum(II) 1,3-d(GTG) intrastrand cross-link, a major adduct in cells treated with the anticancer drug carboplatin.

The platinum 1,3-d(GXG) intrastrand cross-link is one of the adducts formed in the reaction of the antitumor drug cisplatin with DNA, and in fact the major adduct found in cells treated with the cisplatin analogue carboplatin. To determine the 3D structure of this adduct, the duplex d(CTCTGTGTCTC).d(GAGACACAGAG)], where GTG denotes a platinum 1,3-intrastrand cross-link, was prepared and studied with high-resolution (1)H NMR. The solution structure was determined using the SPEDREF protocol, which includes an iterative NOE-restrained refinement procedure. Calculated and recorded NOE spectra were found to be in good agreement (NMR R factor 22%). The studied duplex is more distorted from B-DNA than previously determined structures of the 1,2-d(GG) intrastrand adducts. The base pairing is lost for the 5'G-C and the central T-A base pair in the GTG lesion, and the central thymine is extruded from the minor groove. To accommodate this lesion, the minor groove is widened, and the 5'-guanine ribose adopts an N-type conformation. The helix is unwound locally and is significantly bent toward the major groove. Significant difference between the structural distortion of the 1, 3-d(GTG) cross-link and other Pt-DNA cross-links sheds new light on the observed differences in protein recognition of these lesions, and thus on the possible differences in mechanisms of action of the various Pt-DNA adducts formed in treatment with platinum anticancer complexes.     

Solution structures of aminofluorene [AF]-stacked conformers of the syn [AF]-C8-dG adduct positioned opposite dC or dA at a template-primer junction.

A solution structural study has been undertaken on the aminofluorene-C8-dG ([AF]dG) adduct located at a single-strand-double-strand d(A1-A2-C3-[AF]G4-C5-T6-A7-C8-C9-A10-T11-C12-C13). d(G14-G15-A16-T17-G18-G19-T20- A21-G22-N23) 13/10-mer junction (N = C or A) using proton-proton distance restraints derived from NMR data in combination with intensity-based relaxation matrix refinement computations. This single-strand-double-strand junction models one arm of a replication fork composed of a 13-mer template strand which contains the [AF]dG modification site and a 10-mer primer strand which has been elongated up to the modified guanine with either its complementary dC partner or a dA mismatch. The solution structures establish that the duplex segment retains a minimally perturbed B-DNA conformation with Watson-Crick hydrogen-bonding retained up to the dC5.dG22 base pair. The guanine ring of the [AF]dG4 adduct adopts a syn glycosidic torsion angle and is displaced into the major groove when positioned opposite dC or dA residues. This base displacement of the modified guanine is accompanied by stacking of one face of the aminofluorene ring of [AF]dG4 with the dC5.dG22 base pair, while the other face of the aminofluorene ring is stacked with the purine ring of the nonadjacent dA2 residue. By contrast, the dC and dA residues opposite the junctional [AF]dG4 adduct site adopt distinctly different alignments. The dC23 residue positioned opposite the adduct site is looped out into the minor groove by the aminofluorene ring. The syn displaced orientation of the modified dG with stacking of the aminofluorene and the looped out position of the partner dC could be envisioned to cause polymerase stalling associated with subsequent misalignment leading to frameshift mutations in appropriate sequences. The dA23 residue positioned opposite the adduct site is positioned in the major groove with its purine ring aligned face down over the van der Waals surface of the major groove and its amino group directed toward the T6.A21 base pair. The Hoogsteen edge of the modified guanine of [AF]dG4 and the Watson-Crick edge of dA23 positioned opposite it are approximately coplanar and directed toward each other but are separated by twice the hydrogen-bonding distance required for pairing. This structure of [AF]dG opposite dA at a model template-primer junctional site can be compared with a previous structure of [AF]dG opposite dA within a fully paired duplex [Norman, D., Abuaf, P., Hingerty, B. E., Live, D. , Grunberger, D., Broyde, S., and Patel, D. J. (1989) Biochemistry 28, 7462-7476]. The alignment of the Hoogsteen edge of [AF]dG (syn) positioned opposite the Watson-Crick edge of dA (anti) has been observed for both systems with the separation greater in the case of the junctional alignment in the model template-primer system. However, the aminofluorene ring is positioned in the minor groove in the fully paired duplex while it stacks over the junctional base pair in the template-primer system. This suggests that the syn [AF]dG opposite dA junctional alignment can be readily incorporated within a duplex by a translation of this entity toward the minor groove.     

Stereochemistry modulates the stability of reduced interstrand cross-links arising from R- and S-alpha-CH3-gamma-OH-1,N2-propano-2'-deoxyguanosine in the 5'-CpG-3' DNA sequence

The solution structures of 5'-Cp-N2-dG-3'-R-(alpha)-CH3-propyl-5'-Cp-N2-dG-3' and 5'-Cp-N2-dG-3'-S-(alpha)-CH3-propyl-5'-Cp-N2-dG-3' interstrand DNA cross-links in the 5'-CpG-3' sequence were determined by NMR spectroscopy. These were utilized as chemically stable surrogates for the corresponding carbinolamine interstrand cross-links arising from the crotonaldehyde- and acetaldehyde-derived R- and S-alpha-CH3-gamma-OH-1,N2-propanodeoxyguanosine adducts. The results provide an explanation for the observation that interstrand cross-link formation in the 5'-CpG-3' sequence by the R- and S-alpha-CH3-gamma-OH-1,N2-propanodeoxyguanosine adducts is dependent upon stereochemistry, favoring the R-alpha-CH3-gamma-OH-1,N2-propanodeoxyguanosine adduct [Kozekov, I. D., Nechev, L. V., Moseley, M. S., Harris, C. M., Rizzo, C. J., Stone, M. P., and Harris, T. M. (2003) J. Am. Chem. Soc. 125, 50-61]. Molecular dynamics calculations, restrained by NOE-based distances and empirical restraints, revealed that both the 5'-Cp-N2-dG-3'-R-(alpha)-CH3-propyl-5'-Cp-N2-dG-3' and 5'-Cp-N2-dG-3'-S-(alpha)-CH3-propyl-5'-Cp-N2-dG-3' cross-links were located in the minor groove and retained Watson-Crick hydrogen bonds at the tandem cross-linked C.G base pairs. However, for the 5'-Cp-N2-dG-3'-R-(alpha)-CH3-propyl-5'-Cp-N2-dG-3' cross-link, the (alpha)-CH3 group was positioned in the center of the minor groove, whereas for the 5'-Cp-N2-dG-3'-S-(alpha)-CH3-propyl-5'-Cp-N2-dG-3' cross-link, the (alpha)-CH3 group was positioned in the 3' direction, showing steric interference with the DNA helix. The 5'-Cp-N2-dG-3'-S-(alpha)-CH3-propyl-5'-Cp-N2-dG-3' cross-link exhibited a lower thermal stability as evidenced by NMR spectroscopy as a function of temperature. The two cross-links also exhibited apparent differences in the conformation of the interstrand three-carbon cross-link, which may also contribute to the lower apparent thermodynamic stability of the 5'-Cp-N2-dG-3'-S-(alpha)-CH3-propyl-5'-Cp-N2-dG-3' cross-link.     

NMR evidence for syn-anti interconversion of a trans opened (10R)-dA adduct of benzo[a]pyrene (7S,8R)-diol (9R,10S)-epoxide in a DNA duplex

2D NMR has been used to examine the structure and dynamics of a 12-mer DNA duplex, d(T(1)A(2)G(3)T(4)C(5)A(6)A(7)G(8)G(9)G(10)C(11)A(12))-d(T(13)G(14)C( 15)C(16)C(17)T(18)T(19)G(20)A(21)C(22)T(23)A(24)), containing a 10R adduct at dA(7) that corresponds to trans addition of the N(6)-amino group of dA(7) to (-)-(7S,8R,9R,10S)-7,8-dihydroxy-9, 10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene [(-)-(S,R,R,S)-BP DE-2]. This DNA duplex contains the base sequence for the major dA mutational hot spot in the HPRT gene when Chinese hamster V79 cells are given low doses of the highly carcinogenic (+)-(R,S,S,R)-BP DE-2 enantiomer. NOE data indicate that the hydrocarbon is intercalated on the 5'-side of the modified base as has been seen previously for other oligonucleotides containing BP DE-2 (10R)-dA adducts. 2D chemical exchange-only experiments indicate dynamic behavior near the intercalation site especially at the 10R adducted dA, such that this base interconverts between the normal anti conformation and a less populated syn conformation. Ab initio molecular orbital chemical shift calculations of nucleotide and dinucleotide fragments in the syn and anti conformations support these conclusions. Although this DNA duplex containing a 10R dA adduct exhibits conformational flexibility as described, it is nevertheless more conformationally stable than the corresponding 10S adducted duplex corresponding to trans opening of the carcinogenic isomer (+)-(R,S,S, R)-BP DE-2, which was too dynamic to permit NMR structure determination. UV and imino proton NMR spectral observations indicated pronounced differences between these two diastereomeric 12-mer duplexes, consistent with conformational disorder at the adduct site and/or an equilibrium with a nonintercalated orientation of the hydrocarbon in the duplex containing the 10S adduct. The existence of conformational flexibility around adducts may be related to the occurrence of multiple mutagenic outcomes resulting from a single DE adduct.