Structural basis of accurate replication beyond a bulky major benzo[a]pyrene adduct by human DNA polymerase kappa

Human Y-family DNA polymerase kappa (polκ) is specialized to bypass bulky lesions in DNA in an error-free way, thus protecting cells from carcinogenic bulky DNA adducts. Benzo[a]pyrene (BP) is one of the most ubiquitous polycyclic aromatic hydrocarbons and an environmental carcinogen. BP covalently modifies DNA and generates mutagenic, bulky adducts. The major BP adduct formed in cells is 10S (+)-trans-anti-BP-N²-dG adduct (BP-dG), which is associated with cancer. The molecular mechanism of how polκ replicates BP-dG accurately is not clear. Here we report the structure of polκ captured at the lesion-extension stage: the enzyme is extending the primer strand after the base pair containing the BP-dG adduct in the template strand at the −1 position. Polκ accommodates the BP adduct in the nascent DNA’s minor groove and keeps the adducted DNA helix in a B-form. Two water molecules cover the edge of the minor groove of the replicating base pair (0 position), which is secured by the BP ring in the −1 position in a 5′ orientation. The 5′ oriented BP adduct keeps correct Watson-Crick base pairing in the active site and promotes high fidelity replication. Our structural and biochemical data reveal a unique molecular basis for accurate DNA replication right after the bulky lesion BP-dG.     

Differential DNA recognition and cleavage by EcoRI dependent on the dynamic equilibrium between the two forms of the malondialdehyde-deoxyguanosine adduct

DNA damage may alter the outcome of protein-nucleic acid interactions. The malondialdehyde-deoxyguanosine adduct, 3-(2'-deoxy-beta-d-erythro-pentofuranosyl)pyrimido[1,2-alpha]purin-10-(3H)-one (M(1)dG), miscodes in vivo and in vitro. M(1)dG is an exocyclic adduct that undergoes ring-opening in duplex DNA to form the acyclic adduct, N(2)-(3-oxo-1-propenyl)-deoxyguanosine (N(2)-OPdG). These two adducts have different effects on DNA polymerase bypass and may affect other DNA processing enzymes. We employed the EcoRI restriction endonuclease as a model for the interaction of DNA binding proteins with adducted DNA substrates. The presence of M(1)dG in the EcoRI recognition sequence impaired the ability of the enzyme to cleave DNA, resulting in only 60% cleavage of the adducted strand and 75% cleavage of the complementary strand. Three adducts of similar structure to M(1)dG that are unable to ring-open were cleaved poorly, or not at all, by EcoRI. None of the adducts appeared to inactivate or sequester EcoRI. Additional studies with BssHII and PauI confirmed these results and demonstrated a positional effect of M(1)dG on cleavage efficiency. These data suggest dissimilar modes of protein-nucleic acid interactions based on differences in adduct structure. Comparison of the solution structures of DNA adducts and the crystal structure of EcoRI complexed to substrate suggest a model to explain the functional differences.     

Sequence selectivity, a test of the nature of the covalent adduct formed between benzo[a]pyrene and DNA

A theoretical study is presented of the energetic and structural properties of covalent adducts of benzo[a]pyrene and a DNA fragment. Energy optimisation is performed with the use of minimiser with constraints and an advanced semiempirical energy formula. Three types of adducts are studied: an external complex with the benzopyrene located in the DNA minor groove and two types of intercalative complexes with the carcinogen situated on the 3' side and 5' side of the covalently bound guanine. For each of the adducts the effects of DNA base sequence are examined. It is shown that the results for the intercalative complex with the carcinogen situated on the 5' side of the modified guanine correlate with the experimentally determined sequence preference.     

Effects of benzo[a]pyrene adduct stereochemistry on downstream DNA replication in vitro: evidence for different adduct conformations within the active site of DNA polymerase I (Klenow fragment)

The presence of bulky adducts in DNA is known to interfere with DNA replication not only at the site of the lesion but also at positions up to 5 nucleotides past the adduct location. Kinetic studies of primer extension by exonuclease-deficient E. coli DNA polymerase I (Klenow fragment) (KF) when (+)-trans- or (+)-cis-B[a]P-N(2)-dG adducts were positioned in the double-stranded region of the primer-templates showed that both stereoisomers significantly block downstream replication. However the (+)-cis adduct, which causes a stronger inhibition of the nucleotides insertion across from and immediately past the lesion, affected the downstream replication to a much smaller extent than did the (+)-trans adduct, especially when the B[a]P-modified dG was properly paired with a dC. The effects of mismatches across from the adduct and the sequence context surrounding the adduct were also dependent on the stereochemistry of the B[a]P adduct. Thus, the identity of the nucleotide across from the adduct that provided the best downstream replication was different for the (+)-cis and (+)-trans adducts, a factor that might differentially contribute to the mutagenic bypass of these lesions. These findings provide strong direct evidence that the conformations of the (+)-cis and (+)-trans adducts within the active site of KF are significantly different and probably differentially affect the interactions of the polymerase with the minor groove, thereby leading to different replication trends. The stereochemistry of the adduct was also found to differentially affect the sequence-mediated primer-template misalignments, resulting in different consequences during the bypass of the lesion.     

Structural Mechanism of Replication Stalling on a Bulky Amino-Polycyclic Aromatic Hydrocarbon DNA Adduct by a Y Family DNA Polymerase

Polycyclic aromatic hydrocarbons and their nitro derivatives are culprits of the detrimental health effects of environmental pollution. These hydrophobic compounds metabolize to reactive species and attach to DNA producing bulky lesions, such as N-[deoxyguanosine-8-yl]-1-aminopyrene (APG), in genomic DNA. The bulky adducts block DNA replication by high-fidelity polymerases and compromise replication fidelities and efficiencies by specialized lesion bypass polymerases. Here we present three crystal structures of the DNA polymerase Dpo4, a model translesion DNA polymerase of the Y family, in complex with APG-lesion-containing DNA in pre-insertion and extension stages. APG is captured in two conformations in the pre-insertion complex; one is highly exposed to the solvent, whereas the other is harbored in a shallow cleft between the finger and unique Y family little finger domain. In contrast, APG is in a single conformation at the extension stage, in which the pyrene ring is sandwiched between the little finger domain and a base from the turning back single-stranded template strand. Strikingly, a nucleotide intercalates the DNA helix to form a quaternary complex with Dpo4, DNA, and an incoming nucleotide, which stabilizes the distorted DNA structure at the extension stage. The unique APG DNA conformations in Dpo4 inhibit DNA translocation through the polymerase active site for APG bypass. We also modeled an insertion complex that illustrates a solvent-exposed pyrene ring contributing to an unstable insertion state. The structural work combined with our lesion replication assays provides a novel structural mechanism on bypass of DNA adducts containing polycyclic aromatic hydrocarbon moieties.     

Effects of DNA adduct structure and sequence context on strand opening of repair intermediates and incision by UvrABC nuclease

DNA damage recognition of nucleotide excision repair (NER) in Escherichia coli is achieved by at least two steps. In the first step, a helical distortion is recognized, which leads to a strand opening at the lesion site. The second step involves the recognition of the type of chemical modification in the single-stranded region of DNA during the processing of the lesions by UvrABC. In the current work, by comparing the efficiencies of UvrABC incision of several types of different DNA adducts, we show that the size and position of the strand opening are dependent on the type of DNA adducts. Optimal incision efficiency for the C8-guanine adducts of 2-aminofluorene (AF) and N-acetyl-2-aminofluorene (AAF) was observed in a bubble of three mismatched nucleotides, whereas the same for C8-guanine adduct of 1-nitropyrene and N(2)-guanine adducts of benzo[a]pyrene diol epoxide (BPDE) was noted in a bubble of six mismatched nucleotides. This suggests that the size of the aromatic ring system of the adduct might influence the extent and number of bases associated with the opened strand region catalyzed by UvrABC. We also showed that the incision efficiency of the AF or AAF adduct was affected by the neighboring DNA sequence context, which, in turn, was the result of differential binding of UvrA to the substrates. The sequence context effect on both incision and binding disappeared when a bubble structure of three bases was introduced at the adduct site. We therefore propose that these effects relate to the initial step of damage recognition of DNA structural distortion. The structure-function relationships in the recognition of the DNA lesions, based on our results, have been discussed.     

Structural and biochemical impact of C8-aryl-guanine adducts within the NarI recognition DNA sequence: influence of aryl ring size on targeted and semi-targeted mutagenicity

Chemical mutagens with an aromatic ring system may be enzymatically transformed to afford aryl radical species that preferentially react at the C8-site of 2′-deoxyguanosine (dG). The resulting carbon-linked C8-aryl-dG adduct possesses altered biophysical and genetic coding properties compared to the precursor nucleoside. Described herein are structural and in vitro mutagenicity studies of a series of fluorescent C8-aryl-dG analogues that differ in aryl ring size and are representative of authentic DNA adducts. These structural mimics have been inserted into a hotspot sequence for frameshift mutations, namely, the reiterated G₃-position of the NarI sequence within 12mer (NarI(12)) and 22mer (NarI(22)) oligonucleotides. In the NarI(12) duplexes, the C8-aryl-dG adducts display a preference for adopting an anti-conformation opposite C, despite the strong syn preference of the free nucleoside. Using the NarI(22) sequence as a template for DNA synthesis in vitro, mutagenicity of the C8-aryl-dG adducts was assayed with representative high-fidelity replicative versus lesion bypass Y-family DNA polymerases, namely, Escherichia coli pol I Klenow fragment exo⁻ (Kf⁻) and Sulfolobus solfataricus P2 DNA polymerase IV (Dpo4). Our experiments provide a basis for a model involving a two-base slippage and subsequent realignment process to relate the miscoding properties of C-linked C8-aryl-dG adducts with their chemical structures.     

Base damage,local sequence context and TP53 mutation hotspots: a molecular dynamics study of benzo[a]pyrene induced DNA distortion and mutability

The mutational pattern for the TP53 tumour suppressor gene in lung tumours differs to other cancer types by having a higher frequency of G:C>T:A transversions. The aetiology of this differing mutation pattern is still unknown. Benzo[a]pyrene,diol epoxide (BPDE) is a potent cigarette smoke carcinogen that forms guanine adducts at TP53 CpG mutation hotspot sites including codons 157, 158, 245, 248 and 273. We performed molecular modelling of BPDE-adducted TP53 duplex sequences to determine the degree of local distortion caused by adducts which could influence the ability of nucleotide excision repair. We show that BPDE adducted codon 157 has greater structural distortion than other TP53 G:C>T:A hotspot sites and that sequence context more distal to adjacent bases must influence local distortion. Using TP53 trinucleotide mutation signatures for lung cancer in smokers and non-smokers we further show that codons 157 and 273 have the highest mutation probability in smokers. Combining this information with adduct structural data we predict that G:C>T:A mutations at codon 157 in lung tumours of smokers are predominantly caused by BPDE. Our results provide insight into how different DNA sequence contexts show variability in DNA distortion at mutagen adduct sites that could compromise DNA repair at well characterized cancer related mutation hotspots.     

Mechanistic investigation of the bypass of a bulky aromatic DNA adduct catalyzed by a Y-family DNA polymerase

3-Nitrobenzanthrone (3-NBA), a nitropolyaromatic hydrocarbon (NitroPAH) pollutant in diesel exhaust, is a potent mutagen and carcinogen. After metabolic activation, the primary metabolites of 3-NBA react with DNA to form dG and dA adducts. One of the three major adducts identified is N-(2′-deoxyguanosin-8-yl)-3-aminobenzanthrone (dG^C8-N-ABA). This bulky adduct likely stalls replicative DNA polymerases but can be traversed by lesion bypass polymerases in vivo. Here, we employed running start assays to show that a site-specifically placed dG^C8-N-ABA is bypassed in vitro by Sulfolobus solfataricus DNA polymerase IV (Dpo4), a model Y-family DNA polymerase. However, the nucleotide incorporation rate of Dpo4 was significantly reduced opposite both the lesion and the template position immediately downstream from the lesion site, leading to two strong pause sites. To investigate the kinetic effect of dG^C8-N-ABA on polymerization, we utilized pre-steady-state kinetic methods to determine the kinetic parameters for individual nucleotide incorporations upstream, opposite, and downstream from the dG^C8-N-ABA lesion. Relative to the replication of the corresponding undamaged DNA template, both nucleotide incorporation efficiency and fidelity of Dpo4 were considerably decreased during dG^C8-N-ABA lesion bypass and the subsequent extension step. The lower nucleotide incorporation efficiency caused by the lesion is a result of a significantly reduced dNTP incorporation rate constant and modestly weaker dNTP binding affinity. At both pause sites, nucleotide incorporation followed biphasic kinetics with a fast and a slow phase and their rates varied with nucleotide concentration. In contrast, only the fast phase was observed with undamaged DNA. A kinetic mechanism was proposed for the bypass of dG^C8-N-ABA bypass catalyzed by Dpo4.     

In vitro replication by prokaryotic and eukaryotic polymerases on DNA templates containing site-specific and stereospecific benzo[a]pyrene-7,8-dihydrodiol-9,10-epoxide adducts.

DNA adducts of the environmental carcinogen benzo[a]pyrene-7,8-dihydrodiol-9,10-epoxide (BPDE) interact stereospecifically with prokaryotic and eukaryotic polymerases in vitro. Toward understanding the capacity to replicate past different diastereomers of BPDE at specific sites in DNA, six deoxyoligonucleotides, each 33 bases long, were constructed with stereochemically defined BPDE adducts on adenine N6 at position two of the human N-ras codon 61. Four polymerases that were studied under single encounters with the template-primer complex terminated synthesis one base 3' to the lesion with all the adducted templates. When multiple encounters between polymerase and substrate were permitted, each of the polymerases analyzed revealed a unique pattern for a given adducted template. The general replication pattern was encompassed under two categories, reflecting the significance of the R and S configurations of C10 of the pyrenyl ring attached to the single-stranded DNA template. Furthermore, within each of these categories, every polymerase demonstrated distinct quantitative differences in product accumulation at a given site, for the various adducted templates. Among the polymerases utilized in this study, exonuclease-deficient Klenow fragment of polymerase I (exo- KF) exhibited the most efficient translesion synthesis resulting in approximately 16% full-length products with the modified templates bearing adducts with C10-S configuration. In contrast, chain elongation with bacteriophage T4 DNA polymerase bearing an active 3'-->5' exonucleolytic activity was most strongly inhibited by all six BPDE-adducted templates. Misincorporation of A opposite the adduct occurred in all the templates when polymerized with Sequenase, whereas exo- KF preferentially incorporated C opposite the C10-R BPDE adducts and A opposite the C10-S BPDE adducts.     

Replication past a trans-4-hydroxynonenal minor-groove adduct by the sequential action of human DNA polymerases iota and kappa

The X-ray crystal structure of human DNA polymerase iota (Poliota) has shown that it differs from all known Pols in its dependence upon Hoogsteen base pairing for synthesizing DNA. Hoogsteen base pairing provides an elegant mechanism for synthesizing DNA opposite minor-groove adducts that present a severe block to synthesis by replicative DNA polymerases. Germane to this problem, a variety of DNA adducts form at the N2 minor-groove position of guanine. Previously, we have shown that proficient and error-free replication through the gamma-HOPdG (gamma-hydroxy-1,N2-propano-2'-deoxyguanosine) adduct, which is formed from the reaction of acrolein with the N2 of guanine, is mediated by the sequential action of human Poliota and Polkappa, in which Poliota incorporates the nucleotide opposite the lesion site and Polkappa carries out the subsequent extension reaction. To test the general applicability of these observations to other adducts formed at the N2 position of guanine, here we examine the proficiency of human Poliota and Polkappa to synthesize past stereoisomers of trans-4-hydroxy-2-nonenal-deoxyguanosine (HNE-dG). Even though HNE- and acrolein-modified dGs share common structural features, due to their increased size and other structural differences, HNE adducts are potentially more blocking for replication than gamma-HOPdG. We show here that the sequential action of Poliota and Polkappa promotes efficient and error-free synthesis through the HNE-dG adducts, in which Poliota incorporates the nucleotide opposite the lesion site and Polkappa performs the extension reaction.     

Flanking bases influence the nature of DNA distortion by platinum 1,2-intrastrand (GG) cross-links

The differences in efficacy and molecular mechanisms of platinum anti-cancer drugs cisplatin (CP) and oxaliplatin (OX) are thought to be partially due to the differences in the DNA conformations of the CP and OX adducts that form on adjacent guanines on DNA, which in turn influence the binding of damage-recognition proteins that control downstream effects of the adducts. Here we report a comprehensive comparison of the structural distortion of DNA caused by CP and OX adducts in the TGGT sequence context using nuclear magnetic resonance (NMR) spectroscopy and molecular dynamics (MD) simulations. When compared to our previous studies in other sequence contexts, these structural studies help us understand the effect of the sequence context on the conformation of Pt-GG DNA adducts. We find that both the sequence context and the type of Pt-GG DNA adduct (CP vs. OX) play an important role in the conformation and the conformational dynamics of Pt-DNA adducts, possibly explaining their influence on the ability of many damage-recognition proteins to bind to Pt-DNA adducts.     

Molecular modeling of the major benzo[a]pyrene N2-dG adduct in cases where mutagenesis results are known in double stranded DNA

The potent mutagen/carcinogen benzo[a]pyrene (B[a]P) is metabolically activated to (+)-anti-B[a]PDE, which induces a full spectrum of mutations (e.g. GC-->TA, GC-->AT, etc.). One hypothesis for this complexity is that different mutations are induced by different conformations of its major adduct [+ta]-B[a]P-N2-dG when bypassed during DNA replication (probably by different DNA polymerases). Previous molecular modeling studies suggested that B[a]P-N2-dG adducts can in principle adopt at least 16 potential conformational classes in ds-DNA. Herein we report on molecular modeling studies with the eight conformations most likely to be relevant to base substitution mutagenesis in 10 cases where mutagenesis has been studied in ds-DNA plasmids in E. coli with B[a]P-N2-dG adducts of differing stereoisomers and DNA sequence contexts, as well as in five cases where the conformation is known by NMR. Of the approximately 11,000 structures generated in this study, the computed lowest energy structures are reported for 120 cases (i.e. eight conformations and 15 examples), and their conformations compared. Of the eight conformations, four are virtually always computed to be high in energy. The remaining four lower energy conformations include two with the BP moiety in the minor groove (designated: BPmi5 and BPmi3), and two base-displaced conformations, one with the dG moiety in the major groove (designated: Gma5) and one with the dG in the minor groove (designated: Gmi3). Interestingly, these four are the only conformations that have been observed for B[a]P-N2-dG adducts in NMR studies. Independent of sequence contexts and adduct stereochemistry, BPmi5 structures tend to look reasonably similar, as do BPmi3 structures, while the base-displaced structures Gma5 and BPmi3 tend to show greater variability in structure. A correlation was sought between modeling and mutagenesis results in the case of the low energy conformations BPmi5, BPmi3, Gma5 and Gma3. Plots of log[(G-->T)/(G-->A)] versus energy[(conformation X)-(conformation Y)] were constructed for all six pairwise combinations of these four conformations, and the only plot giving a straight line involved Gma5 and Gmi3. While this finding is striking, its significance is unclear (as discussed).     

Structural basis for the sequence-dependent effects of platinum–DNA adducts

The differences in efficacy and molecular mechanisms of platinum based anti-cancer drugs cisplatin (CP) and oxaliplatin (OX) have been hypothesized to be in part due to the differential binding affinity of cellular and damage recognition proteins to CP and OX adducts formed on adjacent guanines in genomic DNA. HMGB1a in particular exhibits higher binding affinity to CP-GG adducts, and the extent of discrimination between CP- and OX-GG adducts is dependent on the bases flanking the adducts. However, the structural basis for this differential binding is not known. Here, we show that the conformational dynamics of CP- and OX-GG adducts are distinct and depend on the sequence context of the adduct. Molecular dynamics simulations of the Pt-GG adducts in the TGGA sequence context revealed that even though the major conformations of CP- and OX-GG adducts were similar, the minor conformations were distinct. Using the pattern of hydrogen bond formation between the Pt–ammines and the adjacent DNA bases, we identified the major and minor conformations sampled by Pt–DNA. We found that the minor conformations sampled exclusively by the CP-GG adduct exhibit structural properties that favor binding by HMGB1a, which may explain its higher binding affinity to CP-GG adducts, while these conformations are not sampled by OX-GG adducts because of the constraints imposed by its cyclohexane ring, which may explain the negligible binding affinity of HMGB1a for OX-GG adducts in the TGGA sequence context. Based on these results, we postulate that the constraints imposed by the cyclohexane ring of OX affect the DNA conformations explored by OX-GG adduct compared to those of CP-GG adduct, which may influence the binding affinities of HMG-domain proteins for Pt-GG adducts, and that these conformations are further influenced by the DNA sequence context of the Pt-GG adduct.     

Sequence Selectivity,a Test of the Nature of the Covalent Adduct Formed Between Benzo[a]pyrene and DNA

Abstract

A theoretical study is presented of the energetic and structural properties of covalent adducts of benzo[a]pyrene and a DNA fragment. Energy optimisation is performed with the use of minimiser with constraints and an advanced semiempirical energy formula. Three types of adducts are studied: an external complex with the benzopyrene located in the DNA minor groove and two types of intercalative complexes with the carcinogen situated on the 3′side and 5′side of the covalently bound guanine. For each of the adducts the effects of DNA base sequence are examined. It is shown that the results for the intercalative complex with the carcinogen situated on the 5′side of the modified guanine correlate with the experimentally determined sequence preference.     

In vitro DNA and dGMP adducts formation caused by ochratoxin A

Ochratoxin A (OTA), a nephrotoxic and nephrocarcinogenic mycotoxin, leads to the formation of DNA adducts after administration to animals. This could be due to an epigenetic effect. In vitro assays can exclude an indirect effect, where the xenobiotic can generate, in vivo, endogenous reactive compounds which give adducts on DNA. Microsomes prepared from mice or rabbit kidney and liver, used as metabolic activators, were incubated in the presence of commercial salmon testes DNA and OTA, with NADPH or arachidonic acid used as cofactors. Upto 126 DNA adducts for 10(9) nucleotides were detected using the 32P postlabeling method after incubation with the mouse kidney system. Similar results were obtained with rabbit kidney microsomes. Using liver microsomes, the number of DNA adducts detected was much lower. When NADPH was used as a cosubstrate (to explore the cytochrome P450 metabolic pathways), with mice kidney microsomes, the adduct level was only 44% of the one obtained with arachidonic acid. These results lend support to the hypothesis of the preferential activation of OTA by the peroxidase activity of prostaglandin synthases and/or lipoxygenases to direct genotoxic metabolites, and are in agreement with the previously obtained results after in vivo treatment of mice. In order to identify the nucleotides of DNA modified by the OTA metabolites, dAMP, dGMP, dTMP and dCMP were used as substrates under the same conditions as with DNA. The adducts were found only on dGMP. The total adduct level was of 344 adducts per 10(9) nucleotides with the appearance of three major adducts in the presence of arachidonic acid. With NADPH, 271 adducts were obtained per 10(9) nucleotides, with again three major adducts, but only two of them were similar to two adducts obtained in the presence of arachidonic acid. Desferal (desferrioxamine B methanesulphonate), at a 50 microM concentration, did not reduce the adduct level. Adducts were also obtained when polydG, polydC and dG-p-dG were used as alternative substrates, whereas no adducts were obtained with polydA, polydT and polydC. The major adduct obtained after incubation of DNA with OTA, comigrated with the major adduct obtained with dGMP, in two chromatographic solvents. These results show that OTA is metabolized to genotoxic metabolite(s) which interact with the guanine residues of DNA.     

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.     

In vivo detection of a novel endogenous etheno–DNA adduct derived from arachidonic acid and the effects of antioxidants on its formation

Previous studies showed that 7-(1′,2′-dihydroxyheptyl)-substituted etheno DNA adducts are products of reactions with the epoxide of (E)-4-hydroxy-2-nonenal, an oxidation product of ω-6 polyunsaturated fatty acids (PUFAs). In this work, we report the detection of 7-(1′,2′-dihydroxyheptyl)-1,N⁶-ethenodeoxyadenosine (DHHedA) in rodent and human tissues by two independent methods: a ³²P-postlabeling/HPLC method and an isotope dilution liquid chromatography–electrospray ionization–tandem mass spectrometry method, demonstrating for the first time that DHHedA is a background DNA lesion in vivo. We showed that DHHedA can be formed upon incubation of arachidonic acid with deoxyadenosine, supporting the notion that ω-6 PUFAs are the endogenous source of DHHedA formation. Because cyclic adducts are derived from the oxidation of PUFAs, we subsequently examined the effects of antioxidants, α-lipoic acid, Polyphenon E, and vitamin E, on the formation of DHHedA and γ-hydroxy-1,N²-propanodeoxyguanosine (γ-OHPdG), a widely studied acrolein-derived adduct arising from oxidized PUFAs, in the livers of Long Evans Cinnamon (LEC) rats. LEC rats are afflicted with elevated lipid peroxidation and prone to the development of hepatocellular carcinomas. The results showed that although the survival of LEC rats was increased significantly by α-lipoic acid, none of the antioxidants inhibited the formation of DHHedA, and only Polyphenon E decreased the formation of γ-OHPdG. In contrast, vitamin E caused a significant increase in the formation of both γ-OHPdG and DHHedA in the livers of LEC rats.     

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.     

In vitro alkylation of calf thymus DNA by acrylonitrile. Isolation of cyanoethyl-adducts of guanine and thymine and carboxyethyl-adducts of adenine and cytosine

Reaction of the rodent carcinogen acrylonitrile (AN) at pH 5.0 and/or pH 7.0 for 10 and/or 40 days with 2'-deoxyadenosine (dAdo), 2'-deoxycytidine (dCyd), 2'-deoxyguanosine (dGuo), 2'-deoxyinosine (dIno), N6-methyl-2'-deoxyadenosine (N6-Me-dAdo) and thymidine (dThd) resulted in the formation of cyanoethyl and carboxyethyl adducts. Adducts were not detected after 4 h. The adducts isolated were 1-(2-carboxyethyl)-dAdo (1-CE-dAdo), N6-CE-dAdo, 3-CE-dCyd, 7-(2-cyanoethyl)-Gua (7-CNE-Gua), 7,9-bis-CNE-Gua, imidazole ring-opened 7,9-bis-CNE-Gua, 1-CNE-dIno, 1-CE-N6-Me-dAdo and 3-CNE-dThd. Structures were assigned on the basis of UV spectra and electron impact (EI), chemical ionization (CI), desorption chemical ionization (DCI) and Californium-252 fission fragment ionization mass spectra. Evidence is presented which strongly suggests that N6-CE-dAdo was formed by Dimroth rearrangement of 1-CE-dAdo during the reaction between AN and dAdo. The carboxyethyl adducts resulted from initial cyanoethylation (by Michael addition) at a ring nitrogen adjacent to an exocyclic nitrogen atom followed by rapid hydrolysis of the nitrile moiety to a carboxylic acid. It was postulated that the facile hydrolysis is an autocatalyzed reaction resulting from the formation of a cyclic intermediate between nitrile carbon and exocyclic nitrogen. AN was reacted with calf thymus DNA (pH 7.0, 37 degrees C, 40 days) and the relative amounts of adducts isolated were 1-CE-Ade (26%), N6-CE-Ade (8%), 3-CE-Cyt (1%), 7-CNE-Gua (26%), 7,9-bis-CNE-Gua (4%), imidazole ring-opened 7,9-bis-CNE-Gua (19%) and 3-CNE-Thy (16%). Thus a carcinogen once adducted to a base in DNA was shown to be subsequently modified resulting in a mixed pattern of cyanoethylated and carboxyethylated AN-DNA adducts. Three of the adducts (1-CE-Ade, N6-CE-Ade and 3-CE-Cyt) were identical to adducts previously reported by us to be formed following in vitro reaction of the carcinogen beta-propiolactone (BPL) and calf thymus DNA. The results demonstrate that AN can directly alkylate DNA in vitro at a physiological pH and temperature.