Miscoding potential of the N2-ethyl-2'-deoxyguanosine DNA adduct by the exonuclease-free Klenow fragment of Escherichia coli DNA polymerase I

Acetaldehyde, a major metabolite of ethanol, reacts with dG residues in DNA, resulting in the formation of the N(2)-ethyl-2'-deoxyguanosine (N(2)-Et-dG) adduct. This adduct has been detected in lymphocyte DNA of alcohol abusers. To explore the miscoding property of the N(2)-Et-dG DNA adduct, phosphoramidite chemical synthesis was used to prepare site-specifically modified oligodeoxynucleotides containing a single N(2)-Et-dG. These N(2)-Et-dG-modified oligodeoxynucleotides were used as templates for primer extension reactions catalyzed by the 3' --> 5' exonuclease-free (exo(-)) Klenow fragment of Escherichia coli DNA polymerase I. The primer extension was retarded one base prior to the N(2)-Et-dG lesion and opposite the lesion; however, when the enzyme was incubated for a longer time or with increased amounts of this enzyme, full extension occurred. Quantitative analysis of the fully extended products showed the preferential incorporation of dGMP and dCMP opposite the N(2)-Et-dG lesion, accompanied by a small amounts of dAMP and dTMP incorporation and one- and two-base deletions. Steady-state kinetic studies were also performed to determine the frequency of nucleotide insertion opposite the N(2)-Et-dG lesion and chain extension from the 3' terminus from the dN.N(2)-Et-dG (N is C, A, G, or T) pairs. These results indicate that the N(2)-Et-dG DNA adduct may generate G --> C transversions in living cells. Such a mutational spectrum has not been detected with other methylated dG adducts, including 8-methyl-2'-deoxyguanosine, O(6)-methyl-2'-deoxyguanosine, and N(2)-methyl-2'-deoxyguanosine. In addition, N(2)-ethyl-2'-deoxyguanosine triphosphate (N(2)-Et-dGTP) was efficiently incorporated opposite a template dC during DNA synthesis catalyzed by the exo(-) Klenow fragment. The utilization of N(2)-Et-dGTP was also determined by steady-state kinetic studies. N(2)-Et-dG DNA adducts are also formed by the incorporation of N(2)-Et-dGTP into DNA and may cause mutations, leading to the development of alcohol- and acetaldehyde-induced human cancers.     

Basis of Miscoding of the DNA Adduct N2,3-Ethenoguanine by Human Y-family DNA Polymerases

N²,3-Ethenoguanine (N²,3-ϵG) is one of the exocyclic DNA adducts produced by endogenous processes (e.g. lipid peroxidation) and exposure to bioactivated vinyl monomers such as vinyl chloride, which is a known human carcinogen. Existing studies exploring the miscoding potential of this lesion are quite indirect because of the lability of the glycosidic bond. We utilized a 2′-fluoro isostere approach to stabilize this lesion and synthesized oligonucleotides containing 2′-fluoro-N²,3-ϵ-2′-deoxyarabinoguanosine to investigate the miscoding potential of N²,3-ϵG by Y-family human DNA polymerases (pols). In primer extension assays, pol η and pol κ replicated through N²,3-ϵG, whereas pol ι and REV1 yielded only 1-base incorporation. Steady-state kinetics revealed that dCTP incorporation is preferred opposite N²,3-ϵG with relative efficiencies in the order of pol κ > REV1 > pol η ≈ pol ι, and dTTP misincorporation is the major miscoding event by all four Y-family human DNA pols. Pol ι had the highest dTTP misincorporation frequency (0.71) followed by pol η (0.63). REV1 misincorporated dTTP and dGTP with much lower frequencies. Crystal structures of pol ι with N²,3-ϵG paired to dCTP and dTTP revealed Hoogsteen-like base pairing mechanisms. Two hydrogen bonds were observed in the N²,3-ϵG:dCTP base pair, whereas only one appears to be present in the case of the N²,3-ϵG:dTTP pair. Base pairing mechanisms derived from the crystal structures explain the slightly favored dCTP insertion for pol ι in steady-state kinetic analysis. Taken together, these results provide a basis for the mutagenic potential of N²,3-ϵG.     

Mechanism of translesion synthesis past an equine estrogen-DNA adduct by Y-family DNA polymerases

4-Hydroxyequilenin (4-OHEN)-dC is a major, potentially mutagenic DNA adduct induced by equine estrogens used for hormone replacement therapy. To study the miscoding property of 4-OHEN-dC and the involvement of Y-family human DNA polymerases (pols) eta, kappa and iota in that process, we incorporated 4-OHEN-dC into oligodeoxynucleotides and used them as templates in primer extension reactions catalyzed by pol eta, kappa and iota. Pol eta inserted dAMP opposite 4-OHEN-dC, accompanied by lesser amounts of dCMP and dTMP incorporation and base deletion. Pol kappa promoted base deletions as well as direct incorporation of dAMP and dCMP. Pol iota worked in conjunction with pol kappa, but not with pol eta, at a replication fork stalled by the adduct, resulting in increased dTMP incorporation. Our results provide a direct evidence that Y-family DNA pols can switch with one another during synthesis past the lesion. No direct incorporation of dGMP, the correct base, was observed with Y-family enzymes. The miscoding potency of 4-OHEN-dC may be associated with the development of reproductive cancers observed in women receiving hormone replacement therapy.     

Miscoding properties of 1,N6-ethanoadenine, a DNA adduct derived from reaction with the antitumor agent 1,3-bis(2-chloroethyl)-1-nitrosourea

1,N(6)-Ethanoadenine (EA) is an exocyclic adduct formed from DNA reaction with the antitumor agent, 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU). To understand the role of this adduct in the mechanism of mutagenicity or carcinogenicity by BCNU, an oligonucleotide with a site-specific EA was synthesized using phosphoramidite chemistry. We now report the in vitro miscoding properties of EA in translesion DNA synthesis catalyzed by mammalian DNA polymerases (pols) alpha, beta, eta and iota. These data were also compared with those obtained for the structurally related exocyclic adduct, 1,N(6)-ethenoadenine (epsilonA). Using a primer extension assay, both pols alpha and beta were primarily blocked by EA or epsilonA with very minor extension. Pol eta, a member of the Y family of polymerases, was capable of catalyzing a significant amount of bypass across both adducts. Pol eta incorporated all four nucleotides opposite EA and epsilonA, but with differential preferences and mainly in an error-prone manner. Human pol iota, a paralog of human pol eta, was blocked by both adducts with a very small amount of synthesis past epsilonA. It incorporated C and, to a much lesser extent, T, opposite either adduct. In addition, the presence of an A adduct, e.g. epsilonA, could affect the specificity of pol iota toward the template T immediately 3' to the adduct. In conclusion, the four polymerases assayed on templates containing an EA or epsilonA showed differential bypass capacity and nucleotide incorporation specificity, with the two adducts not completely identical in influencing these properties. Although there was a measurable extent of error-free nucleotide incorporation, all these polymerases primarily misincorporated opposite EA, indicating that the adduct, similar to epsilonA, is a miscoding lesion.     

Quantitative analysis of the mutagenic potential of 1-aminopyrene-DNA adduct bypass catalyzed by Y-family DNA polymerases

N-(Deoxyguanosin-8-yl)-1-aminopyrene (dG(AP)) is the predominant nitro polyaromatic hydrocarbon product generated from the air pollutant 1-nitropyrene reacting with DNA. Previous studies have shown that dG(AP) induces genetic mutations in bacterial and mammalian cells. One potential source of these mutations is the error-prone bypass of dG(AP) lesions catalyzed by the low-fidelity Y-family DNA polymerases. To provide a comparative analysis of the mutagenic potential of the translesion DNA synthesis (TLS) of dG(AP), we employed short oligonucleotide sequencing assays (SOSAs) with the model Y-family DNA polymerase from Sulfolobus solfataricus, DNA Polymerase IV (Dpo4), and the human Y-family DNA polymerases eta (hPolη), kappa (hPolκ), and iota (hPolι). Relative to undamaged DNA, all four enzymes generated far more mutations (base deletions, insertions, and substitutions) with a DNA template containing a site-specifically placed dG(AP). Opposite dG(AP) and at an immediate downstream template position, the most frequent mutations made by the three human enzymes were base deletions and the most frequent base substitutions were dAs for all enzymes. Based on the SOSA data, Dpo4 was the least error-prone Y-family DNA polymerase among the four enzymes during the TLS of dG(AP). Among the three human Y-family enzymes, hPolκ made the fewest mutations at all template positions except opposite the lesion site. hPolκ was significantly less error-prone than hPolι and hPolη during the extension of dG(AP) bypass products. Interestingly, the most frequent mutations created by hPolι at all template positions were base deletions. Although hRev1, the fourth human Y-family enzyme, could not extend dG(AP) bypass products in our standing start assays, it preferentially incorporated dCTP opposite the bulky lesion. Collectively, these mutagenic profiles suggest that hPolk and hRev1 are the most suitable human Y-family DNA polymerases to perform TLS of dG(AP) in humans.     

Miscoding properties of 2'-deoxyinosine, a nitric oxide-derived DNA Adduct, during translesion synthesis catalyzed by human DNA polymerases

Chronic inflammation involving constant generation of nitric oxide (NO) by macrophages has been recognized as a factor related to carcinogenesis. At the site of inflammation, nitrosatively deaminated DNA adducts such as 2′-deoxyinosine (dI) and 2′-deoxyxanthosine are primarily formed by NO and may be associated with the development of cancer. In this study, we explored the miscoding properties of the dI lesion generated by Y-family DNA polymerases (pols) using a new fluorescent method for analyzing translesion synthesis. An oligodeoxynucleotide containing a single dI lesion was used as a template in primer extension reaction catalyzed by human DNA pols to explore the miscoding potential of the dI adduct. Primer extension reaction catalyzed by pol α was slightly retarded prior to the dI adduct site; most of the primers were extended past the lesion. Pol η and pol κΔC (a truncated form of pol κ) readily bypassed the dI lesion. The fully extended products were analyzed by using two-phased PAGE to quantify the miscoding frequency and specificity occurring at the lesion site. All pols, that is, pol α, pol η, and pol κΔC, promoted preferential incorporation of 2′-deoxycytidine monophosphate (dCMP), the wrong base, opposite the dI lesion. Surprisingly, no incorporation of 2′-deoxythymidine monophosphate, the correct base, was observed opposite the lesion. Steady-state kinetic studies with pol α, pol η, and pol κΔC indicated that dCMP was preferentially incorporated opposite the dI lesion. These pols bypassed the lesion by incorporating dCMP opposite the lesion and extended past the lesion. These relative bypass frequencies past the dC:dI pair were at least 3 orders of magnitude higher than those for the dT:dI pair. Thus, the dI adduct is a highly miscoding lesion capable of generating A → G transition. This NO-induced adduct may play an important role in initiating inflammation-driven carcinogenesis.     

Mutagenesis and repair induced by the DNA advanced glycation end product N2-1-(carboxyethyl)-2'-deoxyguanosine in human cells

Glycation of biopolymers by glucose-derived α-oxo-aldehydes such as methylglyoxal (MG) is believed to play a major role in the complex pathologies associated with diabetes and metabolic disease. In contrast to the extensive literature detailing the formation and physiological consequences of protein glycation, there is little information about the corresponding phenomenon for DNA. To assess the potential contribution of DNA glycation to genetic instability, we prepared shuttle vectors containing defined levels of the DNA glycation adduct N(2)-(1-carboxyethyl)-2'-deoxyguanosine (CEdG) and transfected them into isogenic human fibroblasts that differed solely in the capacity to conduct nucleotide excision repair (NER). In the NER-compromised fibroblasts, the induced mutation frequencies increased up to 18-fold relative to background over a range of ～10-1400 CEdG adducts/10(5) dG, whereas the same substrates transfected into NER-competent cells induced a response that was 5-fold over background at the highest adduct density. The positive linear correlation (R(2) = 0.998) of mutation frequency with increasing CEdG level in NER-defective cells suggested that NER was the primary if not exclusive mechanism for repair of this adduct in human fibroblasts. Consistent with predictions from biochemical studies using CEdG-substituted oligonucleotides, guanine transversions were the predominant mutation resulting from replication of MG-modified plasmids. At high CEdG levels, significant increases in the number of AT → GC transitions were observed exclusively in NER-competent cells (P < 0.0001). This suggested the involvement of an NER-dependent mutagenic process in response to critical levels of DNA damage, possibly mediated by error-prone Y-family polymerases.     

Formation of DNA triple helix containing N(4)-(6-aminopyridin-2-yl)-2'-deoxycytidine.

A cytidinyl derivative, N(4)-(6-aminopyridin-2-yl)- 2'-deoxycytidine ((p)C), could interact with a CG base pair to support the triple-helix (triplex) formation of oligodeoxyribonucleotides. Characteristics of (p)C in the formation of both intramolecular triplex, i.e., a "paper clip type" triplex ((P)CT) and intermolecular triplex, i.e., a "linear type" triplex (LT) was monitored by optical methods and isothermal titration calorimetric measurements. Experimental results revealed that the LT with (p)C*CG internally was independent of the solution pH. Only single substitution of (p)C, situated internally but not terminally, facilitated the (P)CT formation by the UV thermal melting study at the neutral pH. However, the best stabilization of the PCT in acidic conditions occurred when (p)C at the end of the triplex rather than internally. In addition, an LT, but not a (P)CT, containing an alternating (p)CT(p)CT(p)C sequence, could be formed in the conditions of 20 mM MgCl(2) and/or 5 mM spermine. Thus, the presence of several nucleotides of (p)C in proximity along the Hoogsteen strand may lead to structural distortion such that the more flexible LT with multiple substitutions is formed in favor of the more rigid PCT.     

Synthesis of the fully protected phosphoramidite of the benzene-DNA adduct, N2-(4-Hydroxyphenyl)-2'-deoxyguanosine and incorporation of the later into DNA oligomers

N(2)- (4-Hydroxyphenyl)-2'-deoxyguanosine-5'-O-DMT-3'-phosphoramidite has been synthesized and used to incorporate the N(2)-(4-hydroxyphenyl)-2'-dG (N(2)-4-HOPh-dG) into DNA, using solid-state synthesis technology. The key step to obtaining the xenonucleoside is a palladium (Xantphos-chelated) catalyzed N(2)-arylation (Buchwald-Hartwig reaction) of a fully protected 2'-deoxyguanosine derivative by 4-isobutyryloxybromobenzene. The reaction proceeded in good yield and the adduct was converted to the required 5'-O-DMT-3'-O-phosphoramidite by standard methods. The latter was used to synthesize oligodeoxynucleotides in which the N(2)-4-HOPh-dG adduct was incorporated site-specifically. The oligomers were purified by reverse-phase HPLC. Enzymatic hydrolysis and HPLC analysis confirmed the presence of this adduct in the oligomers.     

Mutagenic properties of 3-(deoxyguanosin-N2-yl)-2-acetylaminofluorene, a persistent acetylaminofluorene-derived DNA adduct in mammalian cells

The carcinogen 2-acetylaminofluorene is metabolically activated in cells and reacts with DNA to form N-(deoxyguanosin-8-yl)-2-acetylaminofluorene (dG-C8-AAF), N-(deoxyguanosin-8-yl)-2-aminofluorene (dG-C8-AF), and 3-(deoxyguanosin-N(2)()-yl)-2-acetylaminofluorene (dG-N(2)-AAF) DNA adducts. The dG-N(2)-AAF adduct is the least abundant of the three isomers, but it persists in the tissues of animals treated with this carcinogen. The miscoding and mutagenic properties of dG-C8-AAF and dG-C8-AF have been established; these adducts are readily excised by DNA repair enzymes engaged in nucleotide excision repair. In the present study, oligodeoxynucleotides modified site-specifically with dG-N(2)-AAF were used as DNA templates in primer extension reactions catalyzed by mammalian DNA polymerases. Reactions catalyzed by pol alpha were strongly blocked at a position one base before dG-N(2)-AAF and also opposite this lesion. In contrast, during translesion synthesis catalyzed by pol eta or pol kappa nucleotides were incorporated opposite the lesion. Both pol eta and pol kappa incorporated dCMP, the correct base, opposite dG-N(2)-AAF. In reactions catalyzed by pol eta, small amounts of dAMP misincorporation and one-base deletions were detected at the lesion site. With pol kappa, significant dTMP misincorporation was observed opposite the lesion. Steady-state kinetic analysis confirmed the results obtained from primer extension studies. Single-stranded shuttle vectors containing (5)(')TCCTCCTCXCCTCTC (X = dG-N(2)-AAF, dG-C8-AAF, or dG) were used to establish the frequency and specificity of dG-N(2)-AAF-induced mutations in simian kidney (COS-7) cells. Both lesions promote G --> T transversions overall, with dG-N(2)-AAF being less mutagenic than dG-C8-AAF (3.4% vs 12.5%). We conclude from this study that dG-N(2)-AAF, by virtue of its persistence in tissues, contributes significantly to the mutational spectra observed in AAF-induced mutagenesis and that pol eta, but not pol kappa, may play a role in this process.     

Mutagenic properties of the 8-amino-2'-deoxyguanosine DNA adduct in mammalian cells.

The DNA adduct 8-amino-2'-deoxyguanosine (8-amino-dG) is found in liver DNA of rats treated with the hepatocarcinogen 2-nitropropane. Site-specifically modified oligodeoxynucleotides were used to explore the mutagenic potential of 8-amino-dG in simian kidney (COS-7) cells. Oligodeoxynucleotides (5'-TCCTCCTX1G2CCTCTC and 5'-TCCTCCTG1X2CCTCTC, X = dG or 8-amino-dG) with the lesion positioned at codon 60 or 61 of the non-coding strand of the human c-Ha- ras1 gene were inserted into single-stranded phagemid vectors and transfected into COS-7 cells. The progeny plasmid obtained was used to transform Escherichia coli DH10B. Transformants were analyzed by oligodeoxynucleotide hybridization and DNA sequencing to establish the mutation frequency and spectrum produced by the modified base. The correct base, dCMP, was incorporated preferentially opposite 8-amino-dG at X1and X2. When 8-amino-dG was at X1, targeted GNH2-->T transversions were detected, along with smaller numbers of GNH2-->A transitions and GNH2-->C transversions. When the adduct was at X2, only GNH2-->T transversions were observed. The frequencies of targeted mutation at X1and X2were 2.7 and 1.7%, respectively. Mutation frequency and mutagenic spectrum were sequence context dependent. In addition, non-targeted G-->T transversions, accompanied by some G-->A transitions, were detected 5' to 8-amino-dG when the lesion was at X2. We conclude that 8-amino-dG is a mutagenic lesion, generating G-->T and G-->C transversions and G-->A transitions in mammalian cells.     

Sensitivity of bacteriophage RB69 DNA polymerase to N2-(p-n-butylphenyl)-2'-deoxyguanosine nucleotides

The response of bacteriophage RB69 DNA polymerase to N2-(p-n-butylphenyl)-2'-deoxyguanosine 5'-triphosphate (BuPdGTP), related nucleotides and non-nucleoside inhibitors was measured and compared to values obtained for the closely related DNA polymerase from bacteriophage T4. Both enzymes showed similar responses to inhibitors in terms of Ki values and the ability to utilize BuPdGTP as a substrate. These results provide the relevance of using the recent crystal structure of RB69 DNA polymerase for analysis of BuPdGTP/B family DNA polymerase interactions.     

Predominance of the 1,N2-propano 2'-deoxyguanosine adduct among 4-hydroxy-2-nonenal-induced DNA lesions

4-Hydroxy-2-nonenal (HNE), one of the main aldehydic compounds released during lipid peroxidation, has been proposed to react with DNA bases in cells. Several classes of DNA lesions involving addition of either HNE or its 2,3-epoxide (epox-HNE) have been identified. In the present work, HPLC associated with tandem mass spectrometry was used to determine the pattern of HNE-induced DNA lesions. First, adducts were quantified within isolated DNA treated with HNE under peroxidizing conditions. The 1,N2-propano-2'-deoxyguanosine adduct of HNE (HNE-dGuo) was found to be the major lesion under all conditions studied. 1,N6-Ethenoadenine and 1,N2-ethenoguanine together with their (1,2-dihydroxyheptyl)-substituted derivatives, which all arise from the reaction of epox-HNE with DNA, were produced in significantly lower yields, even in the presence of 20 mM H2O2. The pyrimidopurinone malondialdehyde-2'-deoxyguanosine adduct was also found to be produced, although in very low yield. Similar results were obtained in cultured human monocytes incubated with HNE, because the HNE-dGuo adduct represented more than 95% of the overall adducts to DNA. In addition, the former lesion was poorly repaired, in contrast to 1,N2-ethenoguanine and, to a lesser extent, 1,N6-ethenoadenine. Altogether, these results suggest than HNE-dGuo may represent the best biomarker of the genotoxic effects of HNE.     

Pre-steady-state kinetic investigation of bypass of a bulky guanine lesion by human Y-family DNA polymerases

3-Nitrobenzanthrone (3-NBA), a byproduct of diesel exhaust, is highly present in the environment and poses a significant health risk. Exposure to 3-NBA results in formation of N-(2′-deoxyguanosin-8-yl)-3-aminobenzanthrone (dG^C8-N-ABA), a bulky DNA lesion that is of particular importance due to its mutagenic and carcinogenic potential. If not repaired or bypassed during genomic replication, dG^C8-N-ABA can stall replication forks, leading to senescence and cell death. Here we used pre-steady-state kinetic methods to determine which of the four human Y-family DNA polymerases (hPolη, hPolκ, hPolι, or hRev1) are able to catalyze translesion synthesis of dG^C8-N-ABA in vitro. Our studies demonstrated that hPolη and hPolκ most efficiently bypassed a site-specifically placed dG^C8-N-ABA lesion, making them good candidates for catalyzing translesion synthesis (TLS) of this bulky lesion in vivo. Consistently, our publication (Biochemistry 53, 5323-31) in 2014 has shown that small interfering RNA-mediated knockdown of hPolη and hPolκ in HEK293T cells significantly reduces the efficiency of TLS of dG^C8-N-ABA. In contrast, hPolι and hRev1 were severely stalled by dG^C8-N-ABA and their potential role in vivo was discussed. Subsequently, we determined the kinetic parameters for correct and incorrect nucleotide incorporation catalyzed by hPolη at various positions upstream, opposite, and downstream from dG^C8-N-ABA. Notably, nucleotide incorporation efficiency and fidelity both decreased significantly during dG^C8-N-ABA bypass and the subsequent extension step, leading to polymerase pausing and error-prone DNA synthesis by hPolη. Furthermore, hPolη displayed nucleotide concentration-dependent biphasic kinetics at the two polymerase pause sites, suggesting that multiple enzyme•DNA complexes likely exist during nucleotide incorporation.     

Quantitative analysis of the efficiency and mutagenic spectra of abasic lesion bypass catalyzed by human Y-family DNA polymerases

Higher eukaryotes encode various Y-family DNA polymerases to perform global DNA lesion bypass. To provide complete mutation spectra for abasic lesion bypass, we employed short oligonucleotide sequencing assays to determine the sequences of abasic lesion bypass products synthesized by human Y-family DNA polymerases eta (hPolη), iota (hPolι) and kappa (hPolκ). The fourth human Y-family DNA polymerase, Rev1, failed to generate full-length lesion bypass products after 3 h. The results indicate that hPolι generates mutations with a frequency from 10 to 80% during each nucleotide incorporation event. In contrast, hPolη is the least error prone, generating the fewest mutations in the vicinity of the abasic lesion and inserting dAMP with a frequency of 67% opposite the abasic site. While the error frequency of hPolκ is intermediate to those of hPolη and hPolι, hPolκ has the highest potential to create frameshift mutations opposite the abasic site. Moreover, the time (t₅₀^bypass) required to bypass 50% of the abasic lesions encountered by hPolη, hPolι and hPolκ was 4.6, 112 and 1 823 s, respectively. These t₅₀^bypass values indicate that, among the enzymes, hPolη has the highest abasic lesion bypass efficiency. Together, our data suggest that hPolη is best suited to perform abasic lesion bypass in vivo.     

Enhancement of camptothecin-induced topoisomerase I cleavage complexes by the acetaldehyde adduct N2-ethyl-2'-deoxyguanosine

The activity of DNA topoisomerase I (Top1), an enzyme that regulates DNA topology, is impacted by DNA structure alterations and by the anticancer alkaloid camptothecin (CPT). Here, we evaluated the effect of the acetaldehyde-derived DNA adduct, N²-ethyl-2′-deoxyguanosine (N²-ethyl-dG), on human Top1 nicking and closing activities. Using purified recombinant Top1, we show that Top1 nicking-closing activity remains unaffected in N²-ethyl-dG adducted oligonucleotides. However, the N²-ethyl-dG adduct enhanced CPT-induced Top1–DNA cleavage complexes depending on the relative position of the N²-ethyl-dG adduct with respect to the Top1 cleavage site. The Top1-mediated DNA religation (closing) was selectively inhibited when the N²-ethyl-dG adduct was present immediately 3′ from the Top1 site (position +1). In addition, when the N²-ethyl-dG adduct was located at the −5 position, CPT enhanced cleavage at an alternate Top1 cleavage site immediately adjacent to the adduct, which was then at position +1 relative to this new alternate Top1 site. Modeling studies suggest that the ethyl group on the N²-ethyl-dG adduct located at the 5′ end of a Top1 site (position +1) sterically blocks the dissociation of CPT from the Top1–DNA complex, thereby inhibiting further the religation (closing) reaction.     

Detection of acetaldehyde derived N(2)-ethyl-2'-deoxyguanosine in human leukocyte DNA following alcohol consumption

Epidemiological studies have shown an association between alcohol (ethanol) consumption and increased cancer risk. The effect of alcohol consumption on the levels and persistence of N(2)-ethylidene-2'-deoxyguanosine (N(2)-ethylidene-dG) formed by acetaldehyde, the oxidative metabolite of ethanol, in human leukocyte DNA was investigated. DNA was isolated from venous blood samples obtained from 30 male non-smoking individuals before consumption of alcohol (0h) and subsequently at 3-5h following the consumption of 150mL of vodka (containing 42% pure ethanol). Additional samples were collected 24h and 48h post-alcohol consumption. The levels of N(2)-ethyl-2'-deoxyguanosine (N(2)-ethyl-dG) in the DNA were determined following reduction of N(2)-ethylidene-dG with sodium cyanoborohydride using a liquid chromatography-tandem mass spectrometry selected reaction monitoring method. A slight time-dependent trend showing an increase and decrease in the levels of N(2)-ethyl-dG was observed following consumption of alcohol compared to time 0h, however, the differences were not statistically significant. The average levels of N(2)-ethyl-dG observed at 0h, 3-5h, 24h and 48h time points following ingestion of alcohol were 34.6±21.9, 35.1±21.0, 36.8±20.7 and 35.6±21.1 per 10(8) 2'-deoxynucleosides, respectively. In conclusion, alcohol consumption that could be encountered under social drinking conditions, does not significantly alter the levels of the acetaldehyde derived DNA adduct, N(2)-ethyl-dG in human leukocyte DNA from healthy individuals.     

Alkylpurine-DNA-N-glycosylase excision of 7-(hydroxymethyl)-1,N6-ethenoadenine, a glycidaldehyde-derived DNA adduct

Glycidaldehyde (GDA) is a bifunctional alkylating agent that has been shown to be mutagenic in vitro and carcinogenic in rodents. However, the molecular mechanism by which it exerts these effects is not established. GDA is capable of forming exocyclic hydroxymethyl-substituted etheno adducts on base residues in vitro. One of them, 7-(hydroxymethyl)-1,N6-ethenoadenine (7-hm-epsilonA), was identified as the principal adduct in mouse skin treated with GDA or a glycidyl ether. In this work, using defined oligonucleotides containing a site-specific 7-hm-epsilonA, the human and mouse alkylpurine-DNA-N-glycosylases (APNGs), responsible for the removal of the analogous 1,N6-ethenoadenine (epsilonA) adduct, are shown to recognize and excise 7-hm-epsilonA. Such an activity can be significantly modulated by both 5' neighboring and opposite sequence contexts. The efficiency of human or mouse APNG for excision of 7-hm-epsilonA is about half that, or similar to the excision of epsilonA, respectively. When human or mouse cell-free extracts were tested, however, the extent of 7-hm-epsilonA excision is dramatically lower than that for epsilonA, suggesting that, in the crude extracts, the APNG activities toward these two adducts are differentially affected. Using cell-free extracts from APNG deficient mice, this enzyme is shown to be the primary glycosylase excising 7-hm-epsilonA. A structural approach, using molecular modeling, was employed to examine how the structure of the 7-hm-epsilonA adduct affects DNA conformation, as compared to the epsilonA adduct. These novel substrate specificities could have both biological and structural implications.     

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.