Synthesis and Characterization of Oligonucleotides Containing a Nitrogen Mustard Formamidopyrimidine Monoadduct of Deoxyguanosine

<i>N</i>⁵-Substituted formamidopyrimidine adducts have been observed from the reaction of dGuo or DNA with aziridine containing electrophiles, including nitrogen mustards. However, the role of substituted Fapy-dGuo adducts in the biological response to nitrogen mustards and related species has not been extensively explored. We have developed chemistry for the site-specific synthesis of oligonucleotides containing an <i>N</i>⁵-nitrogen mustard Fapy-dGuo using the phosphoramidite approach. The lesion was found to be a good substrate for Escherichia coli endonuclease IV and formamidopyrimidine glycosylase

Base-Displaced Intercalated Conformation of the 2‑Amino-3-methylimidazo[4,5‑<i>f</i>]quinoline <i>N</i>²‑dG DNA Adduct Positioned at the Nonreiterated G¹ in the <i>Nar</i>I Restriction Site

The conformation of an <i>N</i>²-dG adduct arising from the heterocyclic amine 2-amino-3-methylimidazo­[4,5-<i>f</i>]­quinoline (IQ), a potent food mutagen, was determined in 5′-d­(C¹T²C³<u>X</u>⁴­G⁵C⁶­G⁷C⁸C⁹­A¹⁰­T¹¹C¹²)-3′:5′-d­(G¹³A¹⁴­T¹⁵G¹⁶­G¹⁷C¹⁸­G¹⁹C²⁰­C²¹G²²­A²³G²⁴)-3′; <u>X</u> = <i>N</i>²-dG-IQ, in which the modified nucleotide <u>X</u>⁴ corresponds to G¹ in the 5′-d­(G¹G²­CG³CC)-3′ <i>Nar</i>I restriction endonuclease site. Circular dichroism (CD) revealed blue shifts relative to the unmodified duplex, consistent with adduct-induced twisting, and a hypochromic effect for the IQ absorbance in the near UV region. NMR revealed that the <i>N</i>²-dG-IQ adduct adopted a base-displaced intercalated conformation in which the modified guanine remained in the <i>anti</i> conformation about the glycosidic bond, the IQ moiety intercalated into the duplex, and the complementary base C²¹ was displaced into the major groove. The processing of the <i>N</i>²-dG-IQ lesion by hpol η is sequence-dependent; when placed at the reiterated G³ position, but not at the G¹ position, this lesion exhibits a propensity for frameshift replication [Choi, J. Y., et al. (2006) <i>J. Biol. Chem</i>., <i>281</i>, 25297–25306]. The structure of the <i>N</i>²-dG-IQ adduct at the nonreiterated G¹ position was compared to that of the same adduct placed at the G³ position [Stavros, K. M., et al. (2014) <i>Nucleic Acids Res.</i>, <i>42</i>, 3450–3463]. CD indicted minimal spectral differences between the G¹ vs G³ <i>N</i>²-dG-IQ adducts. NMR indicated that the <i>N</i>²-dG-IQ adduct exhibited similar base-displaced intercalated conformations at both the G¹ and G³ positions. This result differed as compared to the corresponding C8-dG-IQ adducts placed at the same positions. The C8-dG-IQ adduct adopted a minor groove conformation when placed at position G¹ but a base-displaced intercalated conformation when placed at position G³ in the <i>Nar</i>I sequence. The present studies suggest that differences in lesion bypass by hpol η may be mediated by differences in the 3′-flanking sequences, perhaps modulating the ability to accommodate transient strand slippage intermediates

Formation of a <i>N</i>²-dG:<i>N</i>²-dG Carbinolamine DNA Cross-link by the <i>trans</i>-4-Hydroxynonenal-Derived (6<i>S</i>,8<i>R</i>,11<i>S</i>) 1,<i>N</i>²-dG Adduct

Michael addition of <i>trans</i>-4-hydroxynonenal (HNE) to deoxyguanosine yields diastereomeric 1,<i>N</i>²-dG adducts in DNA. When placed opposite dC in the 5′-CpG-3′ sequence, the (6<i>S</i>,8<i>R</i>,11<i>S</i>) diastereomer forms a <i>N</i>²-dG:<i>N</i>²-dG interstrand cross-link [Wang, H.; Kozekov, I. D.; Harris, T. M.; Rizzo, C. J. <i>J. Am. Chem. Soc.</i> <b>2003</b>, <i>125</i>, 5687–5700]. We refined its structure in 5′-d(G¹C²T³A⁴G⁵C⁶<u>X</u>⁷A⁸G⁹T¹⁰C¹¹C¹²)-3′·5′-d(G¹³G¹⁴A¹⁵C¹⁶T¹⁷C¹⁸<u>Y</u>¹⁹C²⁰T²¹A²²G²³C²⁴)-3′ [X⁷ is the dG adjacent to the C6 carbon of the cross-link or the α-carbon of the (6<i>S</i>,8<i>R</i>,11<i>S</i>) 1,<i>N</i>²-dG adduct, and Y¹⁹ is the dG adjacent to the C8 carbon of the cross-link or the γ-carbon of the HNE-derived (6<i>S</i>,8<i>R</i>,11<i>S</i>) 1,<i>N</i>²-dG adduct; the cross-link is in the 5′-CpG-3′ sequence]. Introduction of ¹³C at the C8 carbon of the cross-link revealed one ¹³C8→H8 correlation, indicating that the cross-link existed predominantly as a carbinolamine linkage. The H8 proton exhibited NOEs to Y¹⁹ H1′, C²⁰ H1′, and C²⁰ H4′, orienting it toward the complementary strand, consistent with the (6<i>S</i>,8<i>R</i>,11<i>S</i>) configuration. An NOE was also observed between the HNE H11 proton and Y¹⁹ H1′, orienting the former toward the complementary strand. Imine and pyrimidopurinone linkages were excluded by observation of the Y¹⁹ <i>N</i>²H and X⁷ N1H protons, respectively. A strong H8→H11 NOE and no ³<i>J</i>(¹³C→H) coupling for the ¹³C8–O–C11–H11 eliminated the tetrahydrofuran species derived from the (6<i>S</i>,8<i>R</i>,11<i>S</i>) 1,<i>N</i>²-dG adduct. The (6<i>S</i>,8<i>R</i>,11<i>S</i>) carbinolamine linkage and the HNE side chain were located in the minor groove. The X⁷ <i>N</i>² and Y¹⁹ <i>N</i>² atoms were in the gauche conformation with respect to the linkage, maintaining Watson–Crick hydrogen bonds at the cross-linked base pairs. A solvated molecular dynamics simulation indicated that the anti conformation of the hydroxyl group with respect to C6 of the tether minimized steric interaction and predicted hydrogen bonds involving O8H with C²⁰ <i>O</i>² of the 5′-neighbor base pair G⁵·C²⁰ and O11H with C¹⁸ <i>O</i>² of X⁷·C¹⁸. These may, in part, explain the stability of this cross-link and the stereochemical preference for the (6<i>S</i>,8<i>R</i>,11<i>S</i>) configuration

Characterization of the Deoxyguanosine–Lysine Cross-Link of Methylglyoxal

Methylglyoxal is a mutagenic bis-electrophile that is produced endogenously from carbohydrate precursors. Methylglyoxal has been reported to induce DNA–protein cross-links (DPCs) in vitro and in cultured cells. Previous work suggests that these cross-links are formed between guanine and either lysine or cysteine side chains. However, the chemical nature of the methylglyoxal induced DPC have not been determined. We have examined the reaction of methylglyoxal, deoxyguanosine (dGuo), and <i>N</i>^α-acetyllysine (AcLys) and determined the structure of the cross-link to be the <i>N</i>²-ethyl-1-carboxamide with the lysine side chain amino group (<b>1</b>). The cross-link was identified by mass spectrometry and the structure confirmed by comparison to a synthetic sample. Further, the cross-link between methylglyoxal, dGuo, and a peptide (AcAVAGKAGAR) was also characterized. The mechanism of cross-link formation is likely to involve an Amadori rearrangement

Conformational Interconversion of the <i>trans</i>-4-Hydroxynonenal-Derived (6<i>S</i>,8<i>R</i>,11<i>S</i>) 1,<i>N</i>²-Deoxyguanosine Adduct When Mismatched with Deoxyadenosine in DNA

The (6<i>S</i>,8<i>R</i>,11<i>S</i>) 1,<i>N</i>²-HNE-dGuo adduct of <i>trans</i>-4-hydroxynonenal (HNE) was incorporated into the duplex 5′-d(GCTAGC<u>X</u>AGTCC)-3′·5′-d(GGACT<u>A</u>GCTAGC)-3′ [X = (6<i>S</i>,8<i>R</i>,11<i>S</i>) HNE-dG], in which the lesion was mismatched opposite dAdo. The (6<i>S</i>,8<i>R</i>,11<i>S</i>) adduct maintained the ring-closed 1,<i>N</i>²-HNE-dG structure. This was in contrast to when this adduct was correctly paired with dCyd, conditions under which it underwent ring opening and rearrangement to diastereomeric minor groove cyclic hemiacetals [Huang, H., Wang, H., Qi, N., Lloyd, R. S., Harris, T. M., Rizzo, C. J., and Stone, M. P. (2008) J. Am. Chem. Soc. 130, 10898−10906]. The (6<i>S</i>,8<i>R</i>,11<i>S</i>) adduct exhibited a <i>syn</i>/<i>anti</i> conformational equilibrium about the glycosyl bond. The <i>syn</i> conformation was predominant in acidic solution. Structural analysis of the <i>syn</i> conformation revealed that X⁷ formed a distorted base pair with the complementary protonated A¹⁸. The HNE moiety was located in the major groove. Structural perturbations were observed at the neighbor C⁶·G¹⁹ and A⁸·T¹⁷ base pairs. At basic pH, the <i>anti</i> conformation of X⁷ was the major species. The 1,<i>N</i>²-HNE-dG intercalated and displaced the complementary A¹⁸ in the 5′-direction, resulting in a bulge at the X⁷·A¹⁸ base pair. The HNE aliphatic chain was oriented toward the minor groove. The Watson−Crick hydrogen bonding of the neighboring A⁸·T¹⁷ base pair was also disrupted

Structure of the 1,<i>N</i>²-Ethenodeoxyguanosine Adduct Opposite Cytosine in Duplex DNA: Hoogsteen Base Pairing at pH 5.2

The exocyclic 1,<i>N</i>²-ethenodeoxyguanosine (1,<i>N</i>²-ϵdG) adduct, arising from the reaction of vinyl halides and other vinyl monomers, including chloroacetaldehyde, and lipid peroxidation products with dG, was examined at pH 5.2 in the oligodeoxynucleotide duplex 5′-d(CGCATXGAATCC)-3′·5′-d(GGATTCCATGCG)-3′ (X = 1,<i>N</i>²-ϵdG). Previously, X(<i>anti</i>)·C(<i>anti</i>) pairing was established in this duplex, containing the 5′-TXG-3′ sequence context, at pH 8.6 [Shanmugam, G., Goodenough, A. K., Kozekov, I. D., Harris, T. M., Guengerich, F. P., Rizzo, C. J., and Stone, M. P. (2007) Chem. Res. Toxicol. 20, 1601−1611]. At pH 5.2, the 1,<i>N</i>²-ϵdG adduct decreased the thermal stability of the duplex by ∼13 °C. The 1,<i>N</i>²-ϵdG adduct rotated about the glycosyl bond from the <i>anti</i> to the <i>syn</i> conformation. This resulted in the observation of a strong nuclear Overhauser effect (NOE) between the imidazole proton of 1,<i>N</i>²-ϵdG and the anomeric proton of the attached deoxyribose, accompanied by an NOE to the minor groove A²⁰ H2 proton from the complementary strand. The <i>syn</i> conformation of the glycosyl bond at 1,<i>N</i>²-ϵdG placed the exocyclic etheno moiety into the major groove. This resulted in the observation of NOEs between the etheno protons and the major groove protons of the 5′-neighboring thymine. The 1,<i>N</i>²-ϵdG adduct formed a Hoogsteen pair with the complementary cytosine, characterized by downfield shifts of the amino protons of the cytosine complementary to the exocyclic adduct. The pattern of chemical shift perturbations indicated that the lesion introduced a localized structural perturbation involving the modified base pair and its 3′- and 5′-neighbor base pairs. A second conformational equilibrium was observed, in which both the modified base pair and its 3′-neighboring G·C base pair formed tandem Hoogsteen pairs. The results support the conclusion that at neutral pH, in the 5′-TXG-3′ sequence, the 1,<i>N</i>²-ϵdG adduct exists as a blend of conformations in duplex DNA. These involve the interconversion of the glycosyl torsion angle between the <i>anti</i> and the <i>syn</i> conformations, occurring at an intermediate rate on the NMR time scale

Structure of the 1,<i>N</i>²-Etheno-2′-deoxyguanosine Lesion in the 3′-G(εdG)T-5′ Sequence Opposite a One-Base Deletion

The structure of the 1,<i>N</i>²-ethenodeoxyguanosine lesion (1,<i>N</i>²-εdG) has been characterized in 5′-d(CGCAT<u>X</u>GAATCC)-3′·5′-d(GGATTCATGCG)-3′ (X = 1,<i>N</i>²-εdG), in which there is no dC opposite the lesion. This duplex (named the 1-BD duplex) models the product of translesion bypass of 1,<i>N</i>²-εdG by <i>Sulfolobus solfataricus</i> P2 DNA polymerase IV (Dpo4) [Zang, H., Goodenough, A. K., Choi, J. Y., Irimia, A., Loukachevitch, L. V., Kozekov, I. D., Angel, K. C., Rizzo, C. J., Egli, M., and Guengerich, F. P. (2005) <i>J. Biol. Chem. 280</i>, 29750−29764], leading to a one-base deletion. The <i>T</i>_m of this duplex is 6 °C higher than that of the duplex in which dC is present opposite the 1,<i>N</i>²-εdG lesion and 8 °C higher than that of the unmodified 1-BD duplex. Analysis of NOEs between the 1,<i>N</i>²-εdG imidazole and deoxyribose H1′ protons and between the 1,<i>N</i>²-εdG etheno H6 and H7 protons and DNA protons establishes that 1,<i>N</i>²-εdG adopts the <i>anti</i> conformation about the glycosyl bond and that the etheno moiety is accommodated within the helix. The resonances of the 1,<i>N</i>²-εdG H6 and H7 etheno protons shift upfield relative to the monomer 1,<i>N</i>²-εdG, attributed to ring current shielding, consistent with their intrahelical location. NMR data reveal that Watson−Crick base pairing is maintained at both the 5′ and 3′ neighbor base pairs. The structure of the 1-BD duplex has been refined using molecular dynamics calculations restrained by NMR-derived distance and dihedral angle restraints. The increased stability of the 1,<i>N</i>²-εdG lesion in the absence of the complementary dC correlates with the one-base deletion extension product observed during the bypass of the 1,<i>N</i>²-εdG lesion by the Dpo4 polymerase, suggesting that stabilization of this bulged intermediate may be significant with regard to the biological processing of the lesion

1,<i>N</i>²-Etheno-2′-deoxyguanosine Adopts the <i>syn</i> Conformation about the Glycosyl Bond When Mismatched with Deoxyadenosine

The oligodeoxynucleotide 5′-CGCAT<u>X</u>GAATCC-3′·5′-GGATTC<u>A</u>ATGCG-3′ containing 1,<i>N</i>²-etheno-2′-deoxyguanosine (1,<i>N</i>²-εdG) opposite deoxyadenosine (named the 1,<i>N</i>²-εdG·dA duplex) models the mismatched adenine product associated with error-prone bypass of 1,<i>N</i>²-εdG by the <i>Sulfolobus solfataricus</i> P2 DNA polymerase IV (Dpo4) and by <i>Escherichia coli</i> polymerases pol I <i>exo</i>^<i>–</i> and pol II <i>exo</i>^<i>–</i>. At pH 5.2, the <i>T</i>_m of this duplex was increased by 3 °C as compared to the duplex in which the 1,<i>N</i>²-εdG lesion is opposite dC, and it was increased by 2 °C compared to the duplex in which guanine is opposite dA (the dG·dA duplex). A strong NOE between the 1,<i>N</i>²-εdG imidazole proton and the anomeric proton of the attached deoxyribose, accompanied by strong NOEs to the minor groove A²⁰ H2 proton and the mismatched A¹⁹ H2 proton from the complementary strand, establish that 1,<i>N</i>²-εdG rotated about the glycosyl bond from the <i>anti</i> to the <i>syn</i> conformation. The etheno moiety was placed into the major groove. This resulted in NOEs between the etheno protons and T⁵ CH₃. A strong NOE between A²⁰ H2 and A¹⁹ H2 protons established that A¹⁹, opposite to 1,<i>N</i>²-εdG, adopted the <i>anti</i> conformation and was directed toward the helix. The downfield shifts of the A¹⁹ amino protons suggested protonation of dA. Thus, the protonated 1,<i>N</i>²-εdG·dA base pair was stabilized by hydrogen bonds between 1,<i>N</i>²-εdG N1 and A¹⁹ N1H⁺ and between 1,<i>N</i>²-εdG <i>O</i>⁹ and A¹⁹ <i>N</i>⁶H. The broad imino proton resonances for the 5′- and 3′-flanking bases suggested that both neighboring base pairs were perturbed. The increased stability of the 1,<i>N</i>²-εdG·dA base pair, compared to that of the 1,<i>N</i>²-εdG·dC base pair, correlated with the mismatch adenine product observed during the bypass of 1,<i>N</i>²-εdG by the Dpo4 polymerase, suggesting that stabilization of this mismatch may be significant with regard to the biological processing of 1,<i>N</i>²-εdG

Replication Bypass of the <i>trans</i>-4-Hydroxynonenal-Derived (6<i>S</i>,8<i>R</i>,11<i>S</i>)-1,<i>N</i>²-Deoxyguanosine DNA Adduct by the <i>Sulfolobus solfataricus</i> DNA Polymerase IV

<i>trans</i>-4-Hydroxynonenal (HNE) is the major peroxidation product of ω-6 polyunsaturated fatty acids in vivo. Michael addition of the <i>N</i>²-amino group of dGuo to HNE followed by ring closure of N1 onto the aldehyde results in four diastereomeric 1,<i>N</i>²-dGuo (1,<i>N</i>²-HNE-dGuo) adducts. The (6<i>S</i>,8<i>R</i>,11<i>S</i>)-HNE-1,<i>N</i>²-dGuo adduct was incorporated into the 18-mer templates 5′-d­(TCAT<u>X</u>GAATCCTTCCCCC)-3′ and d­(TCAC<u>X</u>GAATCCTTCCCCC)-3′, where X = (6<i>S</i>,8<i>R</i>,11<i>S</i>)-HNE-1,<i>N</i>²-dGuo adduct. These differed in the identity of the template 5′-neighbor base, which was either Thy or Cyt, respectively. Each of these templates was annealed with either a 13-mer primer 5′-d­(GGGGGAAGGATTC)-3′ or a 14-mer primer 5′-d­(GGGGGAAGGATTCC)-3′. The addition of dNTPs to the 13-mer primer allowed analysis of dNTP insertion opposite to the (6<i>S</i>,8<i>R</i>,11<i>S</i>)-HNE-1,<i>N</i>²-dGuo adduct, whereas the 14-mer primer allowed analysis of dNTP extension past a primed (6<i>S</i>,8<i>R</i>,11<i>S</i>)-HNE-1,<i>N</i>²-dGuo:dCyd pair. The <i>Sulfolobus solfataricus</i> P2 DNA polymerase IV (Dpo4) belongs to the Y-family of error-prone polymerases. Replication bypass studies in vitro reveal that this polymerase inserted dNTPs opposite the (6<i>S</i>,8<i>R</i>,11<i>S</i>)-HNE-1,<i>N</i>²-dGuo adduct in a sequence-specific manner. If the template 5′-neighbor base was dCyt, the polymerase inserted primarily dGTP, whereas if the template 5′-neighbor base was dThy, the polymerase inserted primarily dATP. The latter event would predict low levels of Gua → Thy mutations during replication bypass when the template 5′-neighbor base is dThy. When presented with a primed (6<i>S</i>,8<i>R</i>,11<i>S</i>)-HNE-1,<i>N</i>²-dGuo:dCyd pair, the polymerase conducted full-length primer extension. Structures for ternary (Dpo4-DNA-dNTP) complexes with all four template-primers were obtained. For the 18-mer:13-mer template-primers in which the polymerase was confronted with the (6<i>S</i>,8<i>R</i>,11<i>S</i>)-HNE-1,<i>N</i>²-dGuo adduct, the (6<i>S</i>,8<i>R</i>,11<i>S</i>)-1,<i>N</i>²-dGuo lesion remained in the ring-closed conformation at the active site. The incoming dNTP, either dGTP or dATP, was positioned with Watson–Crick pairing opposite the template 5′-neighbor base, dCyt or dThy, respectively. In contrast, for the 18-mer:14-mer template-primers with a primed (6<i>S</i>,8<i>R</i>,11<i>S</i>)-HNE-1,<i>N</i>²-dGuo:dCyd pair, ring opening of the adduct to the corresponding <i>N</i>²-dGuo aldehyde species occurred. This allowed Watson–Crick base pairing at the (6<i>S</i>,8<i>R</i>,11<i>S</i>)-HNE-1,<i>N</i>²-dGuo:dCyd pair

Translesion DNA Synthesis by Human DNA Polymerase η on Templates Containing a Pyrimidopurinone Deoxyguanosine Adduct, 3-(2′-Deoxy-β-d-<i>erythro</i>-pentofuranosyl)pyrimido-[1,2-<i>a</i>]purin-10(3<i>H</i>)-one

M₁dG (3-(2′-deoxy-β-d-<i>erythro</i>-pentofuranosyl)pyrimido[1,2-<i>a</i>]purin-10(3<i>H</i>)-one) lesions are mutagenic in bacterial and mammalian cells, leading to base substitutions (mostly M₁dG to dT and M₁dG to dA) and frameshift mutations. M₁dG is produced endogenously through the reaction of peroxidation products, base propenal or malondialdehyde, with deoxyguanosine residues in DNA. The mutagenicity of M₁dG in <i>Escherichia coli</i> is dependent on the SOS response, specifically the umuC and umuD gene products, suggesting that mutagenic lesion bypass occurs by the action of translesion DNA polymerases, like DNA polymerase V. Bypass of DNA lesions by translesion DNA polymerases is conserved in bacteria, yeast, and mammalian cells. The ability of recombinant human DNA polymerase η to synthesize DNA across from M₁dG was studied. M₁dG partially blocked DNA synthesis by polymerase η. Using steady-state kinetics, we found that insertion of dCTP was the least favored insertion product opposite the M₁dG lesion (800-fold less efficient than opposite dG). Extension from M₁dG·dC was equally as efficient as from control primer-templates (dG·dC). dATP insertion opposite M₁dG was the most favored insertion product (8-fold less efficient than opposite dG), but extension from M₁dG·dA was 20-fold less efficient than dG·dC. The sequences of full-length human DNA polymerase η bypass products of M₁dG were determined by LC-ESI/MS/MS. Bypass products contained incorporation of dA (52%) or dC (16%) opposite M₁dG or −1 frameshifts at the lesion site (31%). Human DNA polymerase η bypass may lead to M₁dG to dT and frameshift but likely not M₁dG to dA mutations during DNA replication