The structurally related exocyclic guanine adducts α-hydroxypropano-dG (α-OH-PdG), γ-hydroxypropano-dG (γ-OH-PdG), and M1dG are formed when DNA is exposed to the reactive aldehydes acrolein and malondialdehyde (MDA). These lesions are believed to form the basis for the observed cytotoxicity and mutagenicity of acrolein and MDA. In an effort to understand the enzymatic pathways and chemical mechanisms that are involved in the repair of acrolein- and MDA-induced DNA damage, we investigated the ability of the DNA repair enzyme AlkB, an α-ketoglutarate/Fe(II) dependent dioxygenase, to process α-OH-PdG, γ-OH-PdG, and M1dG in both single- and double-stranded DNA contexts. By monitoring the repair reactions using quadrupole time-of-flight (Q-TOF) mass spectrometry, it was established that AlkB can oxidatively dealkylate γ-OH-PdG most efficiently, followed by M1dG and α-OH-PdG. The AlkB repair mechanism involved multiple intermediates and complex, overlapping repair pathways. For example, the three exocyclic guanine adducts were shown to be in equilibrium with open-ring aldehydic forms, which were trapped using (pentafluorobenzyl)hydroxylamine (PFBHA) or NaBH4. AlkB repaired the trapped open-ring form of γ-OH-PdG but not the trapped open-ring of α-OH-PdG. Taken together, this study provides a detailed mechanism by which three-carbon bridge exocyclic guanine adducts can be processed by AlkB and suggests an important role for the AlkB family of dioxygenases in protecting against the deleterious biological consequences of acrolein and MDA.

Acrolein, a mutagenic aldehyde, reacts with deoxyguanosine (dG) to form 3-(2'-deoxy-β-d-erythro-pentofuranosyl)-5,6,7,8-tetrahydro-8-hydroxypyrimido[1,2-a] purin-10(3H)-one (γ-OH-PdG). When placed opposite deoxycytosine (dC) in DNA, γ-OH-PdG undergoes ring-opening to the N(2)-(3-oxopropyl)-dG. Ring-opening of the adduct has been hypothesized to facilitate nonmutagenic bypass, particularly by DNA polymerases of the Y family. This study examined the bypass of γ-OH-PdG by Sulfolobus solfataricus Dpo4, the prototypic Y-family DNA polymerase, using templates that contained the adduct in either the 5'-CXG-3' or the 5'-TXG-3' sequence context. Although γ-OH-PdG partially blocked Dpo4-catalyzed DNA synthesis, full primer extension was observed, and the majority of bypass products were error-free. Conversion of the adduct into an irreversibly ring-opened derivative prior to reaction facilitated bypass and further improved the fidelity. Structures of ternary Dpo4·DNA·dNTP complexes were determined with primers that either were positioned immediately upstream of the lesion (preinsertion complexes) or had a 3'-terminal dC opposite the lesion (postinsertion complexes); the incoming nucleotides, either dGTP or dATP, were complementary to the template 5'-neighbor nucleotide. In both postinsertion complexes, the adduct existed as ring-opened species, and the resulting base-pair featured Watson-Crick hydrogen bonding. The incoming nucleotide paired with the 5'-neighbor template, while the primer 3'-hydroxyl was positioned to facilitate extension. In contrast, γ-OH-PdG was in the ring-closed form in both preinsertion complexes, and the overall structure did not favor catalysis. These data provide insights into γ-OH-PdG chemistry during replication bypass by the Dpo4 DNA polymerase and may explain why γ-OH-PdG-induced mutations due to primer-template misalignment are uncommon.

DNA is prone to attack by physical and chemical agents generated endogenously and exogenously, producing modified DNA bases (i.e. DNA adducts/lesions), abasic sites, and inter- and intrastrand DNA crosslinks. DNA adducts, if not properly repaired, can lead to blocked replication, misincorporation, and mutation, potentially causing gene deregulation and cancer. Etheno (e) DNA adducts are exocyclic adducts that, in addition to their use as fluorescent nucleotidfe derivatives,[1] were first recognized as reaction products of DNA with reactive metabolites of the occupational carcinogen vinyl chloride (VC).[2] Endogenous etheno-DNA adducts, arising from lipid peroxidation-derived DNA damage, were also detected in rats[3] and humans[4] without VC exposure. VC is a known carcinogen that induces hepatic angiosarcomas.[5] The major DNA adduct formed by VC, N7-(2-oxoethyl)guanine,[3, 6] is generally not considered to be mutagenic, because in vitro experiments showed that it did not cause detectable miscoding in an assay with modified poly(GC).[7] However, etheno adducts formed by VC (e.g. 1,N6-ethenoadenine, 3,N4-ethenocytosine, N2,3-ethenoguanine (N2,3-eG), and 1,N2-ethenoguanine (1,N2-eG)) have all been shown to be mutagenic in vitro and in bacteria (see N2,3-eG and 1,N2-eG structures in Figure 1a).[8] N2,3-eG is the most abundant endogenous etheno adduct, with levels estimated to be approximately 36 N2,3-eG lesions/cell in livers of untreated rats or humans.[9] A common assumption is that N2,3-eG is highly mutagenic; N2,3-eG is considered to contribute to the carcinogenesis of VC and inflammation-driven malignancies.[10] The dominance of a GC to AT transition in five of six K-ras (oncogene) tumors from VC workers[9] suggests the importance of a G adduct, but the misincorporation characteristics of 1,N2-eG are not consistent with this transition.[8a,f] Little repair of N2,3-eG occurs in VC-exposed rats, since the half-life of this lesion in rat liver and lung (150 days) and in rat kidney (75 days) is quite long.[11] The lability of the glycosidic bond of N2,3-e-deoxyguanosine (N2,3-e-dG) makes it difficult to adequately discern its mutagenic potential.[12] Both C and T were incorporated opposite N2,3-eG in a polyribo(G/N2,3-eG) template by avian myeloblastosis virus (AMV) reverse transcriptase.[13] N2,3-e-Deoxyguanosine triphosphate is inserted opposite T by several polymerases (pols).[8d]

The oligodeoxynucleotide 5'-CGCATXGAATCC-3'·5'-GGATTCAATGCG-3' containing 1,N(2)-etheno-2'-deoxyguanosine (1,N(2)-εdG) opposite deoxyadenosine (named the 1,N(2)-εdG·dA duplex) models the mismatched adenine product associated with error-prone bypass of 1,N(2)-εdG by the Sulfolobus solfataricus P2 DNA polymerase IV (Dpo4) and by Escherichia coli polymerases pol I exo(-) and pol II exo(-). At pH 5.2, the T(m) of this duplex was increased by 3 °C as compared to the duplex in which the 1,N(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,N(2)-εdG imidazole proton and the anomeric proton of the attached deoxyribose, accompanied by strong NOEs to the minor groove A(20) H2 proton and the mismatched A(19) H2 proton from the complementary strand, establish that 1,N(2)-εdG rotated about the glycosyl bond from the anti to the syn conformation. The etheno moiety was placed into the major groove. This resulted in NOEs between the etheno protons and T(5) CH(3). A strong NOE between A(20) H2 and A(19) H2 protons established that A(19), opposite to 1,N(2)-εdG, adopted the anti conformation and was directed toward the helix. The downfield shifts of the A(19) amino protons suggested protonation of dA. Thus, the protonated 1,N(2)-εdG·dA base pair was stabilized by hydrogen bonds between 1,N(2)-εdG N1 and A(19) N1H(+) and between 1,N(2)-εdG O(9) and A(19)N(6)H. 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,N(2)-εdG·dA base pair, compared to that of the 1,N(2)-εdG·dC base pair, correlated with the mismatch adenine product observed during the bypass of 1,N(2)-εdG by the Dpo4 polymerase, suggesting that stabilization of this mismatch may be significant with regard to the biological processing of 1,N(2)-εdG.

Benzene is employed in large quantities in the chemical industry and is an ubiquitous contaminant in the environment. There is strong epidemiological evidence that benzene exposure induces hematopoietic malignancies, especially acute myeloid leukemia, in humans, but the chemical mechanisms remain obscure. E,E-Muconaldehyde is one of the products of metabolic oxidation of benzene. This paper explores the proposition that E,E-muconaldehyde is capable of forming Gua-Gua cross-links. If formed in DNA, the replication and repair of such cross-links might introduce structural defects that could be the origin of the carcinogenicity. We have investigated the reaction of E,E-muconaldehyde with dGuo and found that the reaction yields two pairs of interconverting diastereomers of a novel heptacyclic bis-adduct having a spiro ring system linking the two Gua residues. The structures of the four diastereomers have been established by NMR spectroscopy and their absolute configurations by comparison of CD spectra with those of model compounds having known configurations. The final two steps in the formation of the bis-nucleoside (5-ring → 6-ring → 7-ring) have significant reversibility, which is the basis for the observed epimerization. The 6-ring precursor was trapped from the equilibrating mixture by reduction with NaBH4. The anti relationship of the two Gua residues in the heptacyclic bis-adduct precludes it from being formed in B DNA, but the 6-ring precursor could readily be accommodated as an interchain or intrachain cross-link. It should be possible to form similar cross-links of dCyt, dAdo, the ε-amino group of lysine, the imidazole NH of histidine, and N termini of peptides with the dGuo-muconaldehyde monoadduct.

DNA−protein conjugates are potentially repaired via proteolytic digestion to DNA−peptide conjugates. The latter have been modeled with the amino-terminal lysine of the peptide KWKK conjugated via a trimethylene linkage to the N2-dG amine positioned in 5′-d(GCTAGCXAGTCC)-3′·5′-d(GGACTCGCTAGC)-3′ (X = N2-dG−trimethylene link−KWKK). This linkage is a surrogate for the reversible linkage formed by the γ-OH-1,N2-propanodeoxyguanosine (γ-OH-PdG) adduct. This conjugated KWKK stabilizes the DNA. Amino acids K26, W27, K28, and K29 are in the minor groove. The W27 indolyl group does not intercalate into the DNA. The G7 N2 amine and the K26 N-terminal amine nitrogens are in the trans configuration with respect to the Cα or Cγ of the trimethylene tether, respectively. The structure of this DNA−KWKK conjugate is discussed in the context of its biological processing.

The structure of the 1,N2-ethenodeoxyguanosine lesion (1,N2-εdG) has been characterized in 5′-d(CGCATXGAATCC)-3′·5′-d(GGATTCATGCG)-3′ (X = 1,N2-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,N2-εdG by Sulfolobus solfataricus 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) J. Biol. Chem. 280, 29750−29764], leading to a one-base deletion. The Tm of this duplex is 6 °C higher than that of the duplex in which dC is present opposite the 1,N2-εdG lesion and 8 °C higher than that of the unmodified 1-BD duplex. Analysis of NOEs between the 1,N2-εdG imidazole and deoxyribose H1′ protons and between the 1,N2-εdG etheno H6 and H7 protons and DNA protons establishes that 1,N2-εdG adopts the anti conformation about the glycosyl bond and that the etheno moiety is accommodated within the helix. The resonances of the 1,N2-εdG H6 and H7 etheno protons shift upfield relative to the monomer 1,N2-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,N2-εdG lesion in the absence of the complementary dC correlates with the one-base deletion extension product observed during the bypass of the 1,N2-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.

Oligonucleotides were synthesized containing the 7-(2-oxoheptyl)-etheno-dGuo adduct, which is derived from the reaction of dGuo and the lipid peroxidation product 4-oxo-2-nonenal. The in vitro replication of 7-(2-oxoheptyl)-etheno-dGuo by the model Y-family polymerase Sulfolobus solfataricus P2 DNA Polymerase IV (Dpo4) was examined in two sequences. The extension products were sequenced using an improved LC-ESI-MS/MS protocol developed in our laboratories, and the results were compared to that of the 1,N(2)-etheno-dGuo adduct in the same sequence contexts. Both etheno adducts were highly miscoding when situated in 5'-TXG-3' local sequence contexts with <4% of the extension products being derived from error-free bypass. The major extension products resulted from the misinsertion of Ade opposite the adduct and a one-base deletion. The major extension products from replication of the etheno lesions in a 5'-CXG-3' local sequence context were the result of misinsertion of Ade, a one-base deletion, and error-free bypass. Other minor extension products were also identified. The 7-(2-oxoheptyl)-etheno-dGuo lesion resulted in a larger frequency of misinsertion of Ade, whereas the 1,N(2)-etheno-dGuo gave more of the one-base deletion product. Conformational studies of duplex DNA containing the 7-(2-oxoheptyl)-etheno-dGuo in a 5'-TXG-3' sequence context by NMR indicated the presence of a pH-dependent conformational transition, likely involving the glycosyl bond at the adducted guanosine; the pK(a) for this transition was lower than that observed for the 1,N(2)-epsilon-dGuo lesion. However, the 7-(2-oxoheptyl)-etheno-dGuo lesion, the complementary Cyt, and both flanking base pairs remained disordered at all pH values, which is attributed to the presence of the hydrophobic heptyl group of the 7-(2-oxoheptyl)-etheno-dGuo lesion. The altered pK(a) value and the structural disorder at the 7-(2-oxoheptyl)-etheno-dGuo lesion site, as compared to the same sequence containing the 1,N(2)-etheno-dGuo, may contribute to higher frequency of misinsertion of Ade.

Trans −4-Hydroxynonenal (HNE) is a peroxidation product of ω−6 polyunsaturated fatty acids. Michael addition of HNE to deoxyguanosine yields four diastereomeric 1, N 2 -dG adducts. The adduct of (6 S ,8 R ,11 S ) stereochemistry forms interstrand N 2 -dG: N 2 -dG cross-links in the 5′-CpG-3′ sequence. It has been compared with the (6 R ,8 S ,11 R ) adduct, incorporated into 5′-d(GCTAGCXAGTCC)-3' · 5'-d(GGACTCGCTAGC)-3′, containing the 5′-CpG-3′ sequence (X = HNE-dG). Both adducts rearrange in DNA to N 2 -dG aldehydes. These aldehydes exist in equilibrium with diastereomeric cyclic hemiacetals, in which the latter predominate at equilibrium. These cyclic hemiacetals mask the aldehydes, explaining why DNA cross-linking is slow compared to related 1, N 2 -dG adducts formed by acrolein and crotonaldehyde. Both the (6 S ,8 R ,11 S ) and (6 R ,8 S ,11 R ) cyclic hemiacetals are located within the minor groove. However, the (6 S ,8 R ,11 S ) cyclic hemiacetal orients in the 5′-direction, while the (6 R ,8 S ,11 R ) cyclic hemiacetal orients in the 3′-direction. The conformations of the diastereomeric N 2 -dG aldehydes, which are the reactive species involved in DNA cross-link formation, have been calculated using molecular mechanics methods. The (6 S ,8 R ,11 S ) aldehyde orients in the 5′-direction, while the (6 R ,8 S ,11 R ) aldehyde orients in the 3′-direction. This suggests a kinetic basis to explain, in part, why the (6 S ,8 R ,11 S ) HNE adduct forms interchain cross-links in the 5′-CpG-3′ sequence, whereas (6 R ,8 S ,11 R ) HNE adduct does not. The presence of these cross-links in vivo is anticipated to interfere with DNA replication and transcription, thereby contributing to the etiology of human disease. Environ. Mol. Mutagen., 2010. © 2010 Wiley-Liss, Inc.

Deoxyinosine (dI) and deoxyxanthosine (dX) are both formed in DNA at appreciable levels in vivo by deamination of deoxyadenosine (dA) and deoxyguanosine (dG), respectively, and can miscode. Structure-activity relationships for dA pairing have been examined extensively using analogs but relatively few studies have probed the roles of the individual hydrogen-bonding atoms of dG in DNA replication. The replicative bacteriophage T7 DNA polymerase/exonuclease and the translesion DNA polymerase Sulfolobus solfataricus pol IV were used as models to discern the mechanisms of miscoding by DNA polymerases. Removal of the 2-amino group from the template dG (i.e., dI) had little impact on the catalytic efficiency of either polymerase, as judged by either steady-state or pre-steady-state kinetic analysis, although the misincorporation frequency was increased by an order of magnitude. dX was highly miscoding with both polymerases, and incorporation of several bases was observed. The addition of an electronegative fluorine atom at the 2-position of dI lowered the oligonucleotide T(m) and strongly inhibited incorporation of dCTP. The addition of bromine or oxygen (dX) at C2 lowered the T(m) further, strongly inhibited both polymerases, and increased the frequency of misincorporation. Linear activity models show the effects of oxygen (dX) and the halogens at C2 on both DNA polymerases as mainly due to a combination of both steric and electrostatic factors, producing a clash with the paired cytosine O2 atom, as opposed to either bulk or perturbation of purine ring electron density alone.

Acrolein (AC) and 4-hydroxy-2-nonenal (HNE) are endogenous bis-electrophiles that arise from the oxidation of polyunsaturated fatty acids. AC is also found in high concentrations in cigarette smoke and automobile exhaust. These reactive α,β-unsaturated aldehyde (enal) covalently modify nucleic acids, to form exocyclic adducts, where the three-carbon hydroxypropano unit bridges the N1 and N(2) positions of deoxyguanosine (dG). The bifunctional nature of these enals allows them to undergo reaction with a second nucleophilic group and form DNA cross-links. These cross-linked enal adducts are likely to contribute to the genotoxic effects of both AC and HNE. We have developed a sensitive mass spectrometric method to detect cross-linked adducts of these enals in calf thymus DNA (CT DNA) treated with AC or HNE. The AC and HNE cross-linked adducts were measured by the stable isotope dilution method, employing a linear quadrupole ion trap mass spectrometer and consecutive reaction monitoring at the MS(3) or MS(4) scan stage. The lower limit of quantification of the cross-linked adducts is ∼1 adduct per 10(8) DNA bases, when 50 μg of DNA is assayed. The cross-linked adducts occur at levels that are ∼1-2% of the levels of the monomeric 1,N(2)-dG adducts in CT DNA treated with either enal.

DNA polymerase ν (POLN or pol ν) is a newly discovered A family polymerase that generates a high error rate when incorporating nucleotides opposite dG; its translesion DNA synthesis (TLS) capability has only been demonstrated for high fidelity replication bypass of thymine glycol lesions. In the current investigation, we describe a novel TLS substrate specificity of pol ν, demonstrating that it is able to bypass exceptionally large DNA lesions whose linkages are through the DNA major groove. Specifically, pol ν catalyzed efficient and high fidelity TLS past peptides linked to N6-dA via a reduced Schiff base linkage with a γ-hydroxypropano-dA. Additionally, pol ν could bypass DNA interstrand cross-links with linkage between N6-dAs in complementary DNA strands. However, the chemically identical DNA−peptide and DNA interstrand cross-links completely blocked pol ν when they were located in the minor groove via a N2-dG linkage. Furthermore, we showed that pol ν incorporated a nucleotide opposite the 1,N6-etheno-dA (εdA) in an error-free manner and (+)-trans-anti-benzo[a]pyrene-7,8-dihydrodiol 9,10-epoxide-dA [(+)-BPDE-dA] in an error-prone manner, albeit with a greatly reduced capability. Collectively, these data suggest that although pol ν bypass capacity cannot be generalized to all major groove DNA adducts, this polymerase could be involved in TLS when genomic replication is blocked by extremely large major groove DNA lesions. In view of the recent observation that pol ν may have a role in cellular tolerance to DNA cross-linking agents, our findings provide biochemical evidence for the potential functioning of this polymerase in the bypass of some DNA−protein and DNA−DNA cross-links.