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 stereochemistry of trans-4-hydroxynonenal-derived exocyclic 1,N2-2'-deoxyguanosine adducts modulates formation of interstrand cross-links in the 5'-CpG-3' sequence

The trans-4-hydroxynonenal (HNE)-derived exocyclic 1, N(2)-dG adduct with (6S,8R,11S) stereochemistry forms interstrand N(2)-dG-N(2)-dG cross-links in the 5'-CpG-3' DNA sequence context, but the corresponding adduct possessing (6R,8S,11R) stereochemistry does not. Both exist primarily as diastereomeric cyclic hemiacetals when placed into duplex DNA [Huang, H., Wang, H., Qi, N., Kozekova, A., Rizzo, C. J., and Stone, M. P. (2008) J. Am. Chem. Soc. 130, 10898-10906]. To explore the structural basis for this difference, the HNE-derived diastereomeric (6S,8R,11S) and (6R,8S,11R) cyclic hemiacetals were examined with respect to conformation when incorporated into 5'-d(GCTAGC XAGTCC)-3' x 5'-d(GGACTCGCTAGC)-3', containing the 5'-CpX-3' sequence [X = (6S,8R,11S)- or (6R,8S,11R)-HNE-dG]. At neutral pH, both adducts exhibited minimal structural perturbations to the DNA duplex that were localized to the site of the adduction at X(7) x C(18) and its neighboring base pair, A(8) x T(17). Both the (6S,8R,11S) and (6R,8S,11R) cyclic hemiacetals were located within the minor groove of the duplex. However, the respective orientations of the two cyclic hemiacetals within the minor groove were dependent upon (6S) versus (6R) stereochemistry. The (6S,8R,11S) cyclic hemiacetal was oriented in the 5'-direction, while the (6R,8S,11R) cyclic hemiacetal was oriented in the 3'-direction. These cyclic hemiacetals effectively mask the reactive aldehydes necessary for initiation of interstrand cross-link formation. From the refined structures of the two cyclic hemiacetals, the conformations of the corresponding diastereomeric aldehydes were predicted, using molecular mechanics calculations. Potential energy minimizations of the duplexes containing the two diastereomeric aldehydes predicted that the (6S,8R,11S) aldehyde was oriented in the 5'-direction while the (6R,8S,11R) aldehyde was oriented in the 3'-direction. These stereochemical differences in orientation suggest a kinetic basis that explains, in part, why the (6S,8R,11S) stereoisomer forms interchain cross-links in the 5'-CpG-3' sequence whereas the (6R,8S,11R) stereoisomer does not.     

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.     

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 (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.     

Enantioselective synthesis of (R)- and (S)-2-methyl-[3,3,2-2H3] alanines

The synthesis of optically pure (R)- and (S)-2-methyl-[3,3,3-2H3] alanines of biological interest is described. The stereochemistry of the reaction of the lithio derivative of (R)-(-)-2,5-dimethoxy-3-benzyl-3-methyl-3,6-dihydropyrazine with alkyl and deuterated alkyl iodides is discussed. The configuration of the newly formed center of chirality in (R)- and (S)-2-methyl-[3,3,3-2H3] alanines is derived from 1H NMR.     

Influence of the R(61,2)- and S(61,2)-alpha-(N6-adenyl)styrene oxide adducts on the A.C mismatched base pair in an oligodeoxynucleotide containing the human N-ras codon 61.

Conformational studies of R- and S-alpha-(N6-adenyl)styrene oxide adducts mismatched with deoxycytosine at position X6 in d(CGGACXAGAAG).d(CTTCTCGTCCG), incorporating codons 60, 61 (underlined), and 62 of the human N-ras protooncogene, are described. These were the R- and S(61,2)C adducts. The S(61,2)C adduct afforded a stable solution structure, while the R(61,2)C adduct resulted in a disordered structure. Distance restraints for the S(61, 2)C adduct were calculated from NOE data using relaxation matrix analysis. These were incorporated as effective potentials into the total energy equation. The structures were refined using restrained molecular dynamics calculations which incorporated a simulated annealing protocol. The accuracy of the emergent structures was evaluated by complete relaxation matrix methods. The structures refined to an average rms difference of 1.07 A, determined by pairwise analysis. The experimentally determined structure was compared to NOE intensity data using complete relaxation matrix back-calculations, yielding an R1x value of 11.2 x 10(-)2. The phenyl ring of the styrene in the S(61,2)C adduct was in the major groove and remained oriented in the 3'-direction as observed for the corresponding S(61,2) adduct paired with thymine [Feng, B., Zhou, L., Pasarelli, M., Harris, C. M., Harris, T. M., and Stone, M. P. (1995) Biochemistry 34, 14021-14036]. A shift of the modified adenine toward the minor groove resulted in the styrenyl ring stacking with nucleotide C5 on the 5'-side of the lesion, which shifted toward the major groove. Unlike the unmodified A.C mismatch, neither the S(61,2)C nor the R(61,2)C adduct formed protonated wobble A.C hydrogen bonds. This suggests that protonated wobble A.C pairing need not be prerequisite to low levels of alpha-SO-induced A --> G mutations. The shift of the modified adenine toward the minor groove in the S(61,2)C structure may play a more important role in the genesis of A --> G mutations. The disordered structure of the R(61,2)C adduct provides a potential explanation as to why that adduct does not induce A --> G mutations.     

Two-step error-prone bypass of the (+)- and (-)-trans-anti-BPDE-N2-dG adducts by human DNA polymerases eta and kappa

Benzo[a]pyrene is a polycyclic aromatic hydrocarbon (PAH) associated with potent carcinogenic activity. Mutagenesis induced by benzo[a]pyrene DNA adducts is believed to involve error-prone translesion synthesis opposite the lesion. However, the DNA polymerase involved in this process has not been clearly defined in eukaryotes. Here, we provide biochemical evidence suggesting a role for DNA polymerase eta (Poleta) in mutagenesis induced by benzo[a]pyrene DNA adducts in cells. Purified human Poleta predominantly inserted an A opposite a template (+)- and (-)-trans-anti-BPDE-N2-dG, two important DNA adducts of benzo[a]pyrene. Both lesions also dramatically elevated G and T mis-insertion error rates of human Poleta. Error-prone nucleotide insertion by human Poleta was more efficient opposite the (+)-trans-anti-BPDE-N2-dG adduct than opposite the (-)-trans-anti-BPDE-N2-dG. However, translesion synthesis by human Poleta largely stopped opposite the lesion and at one nucleotide downstream of the lesion (+1 extension). The limited extension synthesis of human Poleta from opposite the lesion was strongly affected by the stereochemistry of the trans-anti-BPDE-N2-dG adducts, the nucleotide opposite the lesion, and the sequence context 5' to the lesion. By combining the nucleotide insertion activity of human Poleta and the extension synthesis activity of human Polkappa, effective error-prone lesion bypass was achieved in vitro in response to the (+)- and (-)-trans-anti-BPDE-N2-dG DNA adducts.     

Increased flexibility enhances misincorporation: temperature effects on nucleotide incorporation opposite a bulky carcinogen-DNA adduct by a Y-family DNA polymerase

The Y-family DNA polymerase Dpo4, from the thermophilic crenarchaeon Sulfolobus solfataricus P2, offers a valuable opportunity to investigate the effect of conformational flexibility on the bypass of bulky lesions because of its ability to function efficiently at a wide range of temperatures. Combined molecular modeling and experimental kinetic studies have been carried out for 10S-(+)-trans-anti-[BP]-N2-dG ((+)-ta-[BP]G), a lesion derived from the covalent reaction of a benzo[a]pyrene metabolite with guanine in DNA, at 55 degrees C and results compared with an earlier study at 37 degrees C (Perlow-Poehnelt, R. A., Likhterov, I., Scicchitano, D. A., Geacintov, N. E., and Broyde, S. (2004) J. Biol. Chem. 279, 36951-36961). The experimental results show that there is more overall nucleotide insertion opposite (+)-ta-[BP]G due to particularly enhanced mismatch incorporation at 55 degrees C compared with 37 degrees C. The molecular dynamics simulations suggest that mismatched nucleotide insertion opposite (+)-ta-[BP]G is increased at 55 degrees C compared with 37 degrees C because the higher temperature shifts the preference of the damaged base from the anti to the syn conformation, with the carcinogen on the more open major groove side. The mismatched dNTP structures are less distorted when the damaged base is syn than when it is anti, at the higher temperature. However, with the normal partner dCTP, the anti conformation with close to Watson-Crick alignment remains more favorable. The molecular dynamics simulations are consistent with the kcat values for nucleotide incorporation opposite the lesion studied, providing structural interpretation of the experimental observations. The observed temperature effect suggests that conformational flexibility plays a role in nucleotide incorporation and bypass fidelity opposite (+)-ta-[BP]G by Dpo4.     

The stereochemistry of benzo[a]pyrene-2'-deoxyguanosine adducts affects DNA methylation by SssI and HhaI DNA methyltransferases

The biologically most significant genotoxic metabolite of the environmental pollutant benzo[a]pyrene (B[a]P), (+)-7R,8S-diol 9S,10R-epoxide, reacts chemically with guanine in DNA, resulting in the predominant formation of (+)-trans-B[a]P-N(2)-dG and, to a lesser extent, (+)-cis-B[a]P-N(2)-dG adducts. Here, we compare the effects of the adduct stereochemistry and conformation on the methylation of cytosine catalyzed by two purified prokaryotic DNA methyltransferases (MTases), SssI and HhaI, with the lesions positioned within or adjacent to their CG and GCGC recognition sites, respectively. The fluorescence properties of the pyrenyl residues of the (+)-cis-B[a]P-N(2)-dG and (+)-trans-B[a]P-N(2)-dG adducts in complexes with MTases are enhanced, but to different extents, indicating that aromatic B[a]P residues are positioned in different microenvironments in the DNA-protein complexes. We have previously shown that the (+)-trans-isomeric adduct inhibits both the binding and methylating efficiencies (k(cat)) of both MTases [Subach OM, Baskunov VB, Darii MV, Maltseva DV, Alexandrov DA, Kirsanova OV, Kolbanovskiy A, Kolbanovskiy M, Johnson F, Bonala R, et al. (2006) Biochemistry45, 6142-6159]. Here we show that the stereoisomeric (+)-cis-B[a]P-N(2)-dG lesion has only a minimal effect on the binding of these MTases and on k(cat). The minor-groove (+)-trans adduct interferes with the formation of the normal DNA minor-groove contacts with the catalytic loop of the MTases. However, the intercalated base-displaced (+)-cis adduct does not interfere with the minor-groove DNA-catalytic loop contacts, allowing near-normal binding of the MTases and undiminished k(cat) values.     

Differential incision of bulky carcinogen-DNA adducts by the UvrABC nuclease: comparison of incision rates and the interactions of Uvr subunits with lesions of different structures

The UvrABC nuclease system from Escherichia coli removes DNA damages induced by a wide range of chemical carcinogens with variable efficiencies. The interactions with UvrABC proteins of the following three lesions site-specifically positioned in DNA, and of known conformations, were investigated: (i) adducts derived from the binding of the (-)-(7S,8R,9R,10S) enantiomer of 7,8-dihydroxy-9, 10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene [(-)-anti-BPDE] by cis-covalent addition to N(2)-2'-deoxyguanosine [(-)-cis-anti-BP-N(2)-dG], (ii) an adduct derived from the binding of the (+)-(1R,2S,3S,4R) enantiomer of 1,2-dihydroxy-3,4-epoxy-1,2,3, 4-tetrahydro-5-methylchrysene [(+)-anti-5-MeCDE] by trans addition to N(2)-2'-deoxyguanosine [(+)-trans-anti-MC-N(2)-dG], and (iii) a C8-2'-deoxyguanosine adduct (C8-AP-dG) formed by reductively activated 1-nitropyrene (1-NP). The influence of these three different adducts on UvrA binding affinities, formation of UvrB-DNA complexes by quantitative gel mobility shift analyses, and the rates of UvrABC incision were investigated. The binding affinities of UvrA varied among the three adducts. UvrA bound to the DNA adduct (+)-trans-anti-MC-N(2)-dG with the highest affinity (K(d) = 17 +/- 2 nM) and to the DNA containing C8-AP-dG with the least affinity (K(d) = 28 +/- 1 nM). The extent of complex formation with UvrB was also the lowest with the C8-AP-dG adduct. 5' Incisions occurred at the eighth phosphate from the modified guanine. The major 3' incision site corresponded to the fifth phosphodiester bond for all three adducts. However, additional 3' incisions were observed at the fourth and sixth phosphates in the case of the C8-AP-dG adduct, whereas in the case of the (-)-cis-anti-BP-N(2)-dG and (+)-trans-anti-MC-N(2)-dG lesions additional 3' cleavage occurred at the sixth and seventh phosphodiester bonds. Both the initial rate and the extent of 5' and 3' incisions revealed that C8-AP-dG was repaired less efficiently in comparison to the (-)-cis-anti-BP-N(2)-dG and (+)-trans-anti-MC-N(2)-dG containing DNA adducts. Our study showed that UvrA recognizes conformational changes induced by structurally different lesions and that in certain cases the binding affinities of UvrA and UvrB can be correlated with the incision rates. The size of the bubble formed around the damaged site with mismatched bases also appears to influence the incision rates. A particularly noteworthy finding in this study is that UvrABC repair of a substrate with no base opposite C8-AP-dG was quite inefficient as compared to the same adduct with a C opposite it. These findings are discussed in terms of the available NMR solution structures.     

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.     

Activities of human DNA polymerase kappa in response to the major benzo[a]pyrene DNA adduct: error-free lesion bypass and extension synthesis from opposite the lesion

In cells, the major benzo[a]pyrene DNA adduct is the highly mutagenic (+)-trans-anti-BPDE-N(2)-dG. In eukaryotes, little is known about lesion bypass of this DNA adduct during replication. Here, we show that purified human Polkappa can effectively bypass a template (+)-trans-anti-BPDE-N(2)-dG adduct in an error-free manner. Kinetic parameters indicate that Polkappa bypass of the (-)-trans-anti-BPDE-N(2)-dG adduct was approximately 41-fold more efficient compared to the (+)-trans-anti-BPDE-N(2)-dG adduct. Furthermore, we have found another activity of human Polkappa in response to the (+)- and (-)-trans-anti-BPDE-N(2)-dG adducts: extension synthesis from mispaired primer 3' ends opposite the lesion. In contrast, the two adducts strongly blocked DNA synthesis by the purified human Polbeta and the purified catalytic subunits of yeast Polalpha, Poldelta, and Pol epsilon right before the lesion. Extension by human Polkappa from the primer 3' G opposite the (+)- and (-)-trans-anti-BPDE-N(2)-dG adducts was mediated by a -1 deletion mechanism, probably resulting from re-aligning the primer G to pair with the next template C by Polkappa prior to DNA synthesis. Thus, sequence contexts 5' to the lesion strongly affect the fidelity and mechanism of the Polkappa-catalyzed extension synthesis. These results support a dual-function model of human Polkappa in bypass of BPDE DNA adducts: it may function both as an error-free bypass polymerase alone and an extension synthesis polymerase in combination with another polymerase.     

Nucleotide selectivity opposite a benzo[a]pyrene-derived N2-dG adduct in a Y-family DNA polymerase: a 5'-slippage mechanism

The Y-family DNA polymerase Dpo4, from the archaeon bacterium Sulfolobus solfataricus, is a member of the DinB family, which also contains human Pol kappa. It has a spacious active site that can accommodate two templating bases simultaneously, with one of them skipped by the incoming dNTP. Assays of single dNTP insertion opposite a benzo[ a]pyrene-derived N (2)-dG adduct, 10 S(+)- trans- anti-[BP]- N (2)-dG ([BP]G*), reveal that an incoming dATP is significantly preferred over the other three dNTPs in the TG 1*G 2 sequence context. Molecular modeling and dynamics simulations were carried out to interpret this experimental observation on a molecular level. Modeling studies suggest that the significant preference for dATP insertion observed experimentally can result from two possible dATP incorporation modes. The dATP can be inserted opposite the T on the 5' side of the adduct G 1*, using an unusual 5'-slippage pattern, in which the unadducted G 2, rather than G 1*, is skipped, to produce a -1 deletion. In addition, the dATP can be misincorporated opposite the adduct. The 5'-slippage pattern may be generally facilitated in cases where the base 3' to the lesion is the same as the adducted base.     

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.     

DNA sequence modulates the conformation of the food mutagen 2-amino-3-methylimidazo[4,5-f]quinoline in the recognition sequence of the NarI restriction enzyme

The conformations of C8-dG adducts of 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) positioned in the C-X1-G, G-X2-C, and C-X3-C contexts in the C-G1-G2-C-G3-C-C recognition sequence of the NarI restriction enzyme were compared, using the oligodeoxynucleotides 5'-d(CTCXGCGCCATC)-3'.5'-d(GATGGCGCCGAG)-3', 5'-d(CTCGXCGCCATC)-3'.5'-d(GATGGCGCCGAG)-3', and 5'-d(CTCGGCXCCATC)-3'.5'-d(GATGGCGCCGAG)-3' (X is the C8-dG adduct of IQ). These were the NarIIQ1, NarIIQ2, and NarIIQ3 duplexes, respectively. In each instance, the glycosyl torsion angle chi for the IQ-modified dG was in the syn conformation. The orientations of the IQ moieties were dependent upon the conformations of torsion angles alpha' [N9-C8-N(IQ)-C2(IQ)] and beta' [C8-N(IQ)-C2(IQ)-N3(IQ)], which were monitored by the patterns of 1H NOEs between the IQ moieties and the DNA in the three sequence contexts. The conformational states of IQ torsion angles alpha' and beta' were predicted from the refined structures of the three adducts obtained from restrained molecular dynamics calculations, utilizing simulated annealing protocols. For the NarIIQ1 and NarIIQ2 duplexes, the alpha' torsion angles were predicted to be -176 +/- 8 degrees and -160 +/- 8 degrees , respectively, whereas for the NarIIQ3 duplex, torsion angle alpha' was predicted to be 159 +/- 7 degrees . Likewise, for the NarIIQ1 and NarIIQ2 duplexes, the beta' torsion angles were predicted to be -152 +/- 8 degrees and -164 +/- 7 degrees , respectively, whereas for the NarIIQ3 duplex, torsion angle beta' was predicted to be -23 +/- 8 degrees . Consequently, the conformations of the IQ adduct in the NarIIQ1 and NarIIQ2 duplexes were similar, with the IQ methyl protons and IQ H4 and H5 protons facing outward in the minor groove, whereas in the NarIIQ3 duplex, the IQ methyl protons and the IQ H4 and H5 protons faced into the DNA duplex, facilitating the base-displaced intercalated orientation of the IQ moiety [Wang, F., Elmquist, C. E., Stover, J. S., Rizzo, C. J., and Stone, M. P. (2006) J. Am. Chem. Soc. 128, 10085-10095]. In contrast, for the NarIIQ1 and NarIIQ2 duplexes, the IQ moiety remained in the minor groove. These sequence-dependent differences suggest that base-displaced intercalation of the IQ adduct is favored when both the 5'- and 3'-flanking nucleotides in the complementary strand are guanines. These conformational differences may correlate with sequence-dependent differences in translesion replication.     

Solution structure of a trans-opened (10S)-dA adduct of (+)-(7S,8R,9S,10R)-7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene in a fully complementary DNA duplex: evidence for a major syn conformation

Two-dimensional NMR was used to determine the solution structure of an undecanucleotide duplex, d(CGGTCACGAGG).d(CCTCGTGACCG), in which (+)-(7S,8R,9S,10R)-7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene is covalently bonded to the exocyclic N(6)() amino group of the central deoxyadenosine, dA(6), through trans addition at C10 of the epoxide (to give a 10S adduct). The present study represents the first NMR structure of a benzo[a]pyrene (10S)-dA adduct in DNA with a complementary T opposite the modified dA. Exchangeable and nonexchangeable protons of the modified duplex were assigned by the use of TOCSY (in D(2)O) and NOESY spectra (in H(2)O and D(2)O). Sequential NOEs expected for a B-type DNA conformation with typical Watson-Crick base pairing are observed along the duplex, except at the lesion site. We observed a strong intraresidue NOE cross-peak between H1' and H8 of the modified dA(6). The sugar H2' and H2' ' of dC(5) lacked NOE cross-peaks with H8 of dA(6) but showed weak interactions with H2 of dA(6) instead. In addition, the chemical shift of the H8 proton (7.51 ppm) of dA(6) appears at a higher field than that of H2 (8.48 ppm). These NOE and chemical shift data for the dA(6) base protons are typical of a syn glycosidic bond at the modified base. Restrained molecular dynamics/energy minimization calculations show that the hydrocarbon is intercalated from the major groove on the 3'-side of the modified base between base pairs A(6)-T(17) and C(7)-G(16) and confirm the syn glycosidic angle (58 degrees ) of the modified dA(6). In the syn structure, a weak A-T hydrogen bond is possible between the N3-H proton of T(17) and N7 of dA(6) (at a distance of 3.11 A), whereas N1, the usual hydrogen bonding partner for N3-H of T when dA is in the anti conformation, is 6.31 A away from this proton. The 10(S)-dA modified DNA duplex remains in a right-handed helix, which bends in the direction of the aliphatic ring of BaP at about 42 degrees from the helical axis. ROESY experiments provided evidence for interconversion between the major, syn conformer and a minor, possibly anti, conformer.     

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.     

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.