Mechanisms of accurate translesion synthesis by human DNA polymerase eta

The XPV (xeroderma pigmentosum variant) gene encodes human DNA polymerase eta (pol eta), which is involved in the replication of damaged DNA. Pol eta catalyzes efficient and accurate translesion synthesis past cis-syn cyclobutane di-thymine lesions. Here we show that human pol eta can catalyze translesion synthesis past an abasic (AP) site analog, N-2-acetylaminofluorene (AAF)-modified guanine, and a cisplatin-induced intrastrand cross-link between two guanines. Pol eta preferentially incorporated dAMP and dGMP opposite AP, and dCMP opposite AAF-G and cisplatin-GG, but other nucleotides were also incorporated opposite these lesions. However, after incorporating an incorrect nucleotide opposite a lesion, pol eta could not continue chain elongation. In contrast, after incorporating the correct nucleotide opposite a lesion, pol eta could continue chain elongation, whereas pol alpha could not. Thus, the fidelity of translesion synthesis by human pol eta relies not only on the ability of this enzyme to incorporate the correct nucleotide opposite a lesion, but also on its ability to elongate only DNA chains that have a correctly incorporated nucleotide opposite a lesion.     

Translesional synthesis past acetylaminofluorene-derived DNA adducts catalyzed by human DNA polymerase kappa and Escherichia coli DNA polymerase IV.

Human DNA polymerase kappa (pol kappa) has a sequence significantly homologous with that of Escherichia coli DNA polymerase IV (pol IV). We used a truncated form of human pol kappa (pol kappaDeltaC) and full-length pol IV to explore the miscoding properties of these enzymes. Oligodeoxynucleotides, modified site-specifically with N-(deoxyguanosin-8-yl)-2-acetylaminofluorene (dG-AAF) and N-(deoxyguanosin-8-yl)-2-aminofluorene (dG-AF), were used as DNA templates in primer extension reactions that included all four dNTPs. Reactions catalyzed by pol kappaDeltaC were partially blocked one base prior to dG-AAF or dG-AF, and also opposite both lesions. At higher enzyme concentrations, a significant fraction of primer was extended. Analysis of the fully extended reaction product revealed incorporation of dTMP opposite dG-AAF, accompanied by much smaller amounts of dCMP, dAMP, and dGMP and some one- and two-base deletions. The product terminating 3' to the adduct site contained AMP misincorporated opposite dC. On templates containing dG-AF, dAMP, dTMP, and dCMP were incorporated opposite the lesion in approximately equal amounts, together with some one-base and two-base deletions. Steady-state kinetics analysis confirmed the results obtained from primer extension reactions catalyzed by pol kappa. In contract, primer extension reactions catalyzed by pol IV were blocked effectively by dG-AAF and dG-AF. At high concentrations of pol IV, full-length products were formed containing primarily one- or two-base deletions with dCMP, the correct base, incorporated opposite dG-AF. The miscoding properties of pol kappa observed in this study are consistent with mutational spectra observed when plasmid vectors containing dG-AAF or dG-AF are introduced into simian kidney cells [Shibutani, S., et al. (2001) Biochemistry 40, 3717-3722], supporting a model in which pol kappa plays a role in translesion synthesis past acetylaminofluorene-derived lesions in mammalian cells.     

Translesion synthesis past 2'-deoxyxanthosine, a nitric oxide-derived DNA adduct, by mammalian DNA polymerases

Cellular DNA is damaged by nitric oxide (NO), a multifunctional bioregulator and an environmental pollutant that has been implicated in diseases associated with cancer and chronic inflammation. 2'-Deoxyxanthosine (dX) is a major NO-derived DNA lesion. To explore the mutagenic potential of dX, a 38-mer oligodeoxynucleotide ((5')CATGCTGATGAATTCCTTCXCTTCTTTCCTCTCCCTTT) modified site-specifically with dX at the X position was prepared post-synthetically and used as a DNA template in primer extension reactions catalyzed by calf thymus DNA polymerase (pol) alpha and human DNA pol beta, eta, and kappa. Primer extension reactions catalyzed by pol alpha or beta in the presence of four dNTPs were retarded at the dX lesion while pol eta and kappa readily bypassed the lesion. The fully extended products were analyzed to quantify the miscoding specificity and frequency of dX using two-phase polyacrylamide gel electrophoresis (PAGE). With pol alpha, eta and kappa, incorrect dTMP was preferentially incorporated opposite the lesion, along with lesser amounts of dCMP, the correct base. When pol beta was used, direct incorporation of correct dCMP was primarily observed, accompanied by small amounts of misincorporation of dTMP, dAMP and dGMP. Steady-state kinetic analyses supported the results obtained from the two-phase PAGE assay. dX is a miscoding lesion capable of preferentially generating G-->A mutations. The miscoding frequency varied depending on DNA polymerase used.     

Translesion synthesis past equine estrogen-derived 2'-deoxyadenosine DNA adducts by human DNA polymerases eta and kappa

Hormone replacement therapy (HRT) increases the risk of developing breast, ovarian, and endometrial cancers. Equilin and equilenin are the major components of the widely prescribed drug used for HRT. 4-Hydroxyequilenin (4-OHEN), a major metabolite of equilin and equilenin, promotes 4-OHEN-modified dC, dA, and dG DNA adducts. These DNA adducts were detected in breast tumor and adjacent normal tissues of several patients receiving HRT. We have recently found that the 4-OHEN-dC DNA adduct is a highly miscoding lesion generating C --> T transitions and C --> G transversions. To explore the mutagenic potential of another major 4-OHEN-dA adduct, site-specifically modified oligodeoxynucleotides containing a single diastereoisomer of 4-OHEN-dA (Pk-1, Pk-2, and Pk-3) were prepared by a postsynthetic method and used as DNA templates for primer extension reactions catalyzed by human DNA polymerase (pol) eta and kappa that are highly expressed in the reproductive organs. Primer extension catalyzed by pol eta or pol kappa occurred rapidly on the unmodified template to form fully extended products. With the major 4-OHEN-dA-modified templates (Pk-2 and Pk-3), primer extension was retarded prior to the lesion and opposite the lesion; a fraction of the primers was extended past the lesion. Steady-state kinetic studies with pol eta and pol kappa indicated that dTMP, the correct base, was preferentially incorporated opposite the 4-OHEN-dA lesion. In addition, pol eta and pol kappa bypassed the lesion by incorporating dAMP and dCMP, respectively, opposite the lesion and extended past the lesion. The relative bypass frequency past the 4-OHEN-dA lesion with pol eta was at least 2 orders of magnitude higher than that observed with pol kappa. The bypass frequency past Pk-2 was more efficient than that past Pk-3. Thus, 4-OHEN-dA is a miscoding lesion generating A --> T transversions and A --> G transitions. The miscoding frequency and specificity of 4-OHEN-dA varied depending on the stereoisomer of the 4-OHEN-dA adduct and DNA polymerase used.     

Translesion synthesis past tamoxifen-derived DNA adducts by human DNA polymerases eta and kappa

The long-term treatment of tamoxifen (TAM), widely used for adjuvant chemotherapy and chemoprevention for breast cancer, increases a risk of developing endometrial cancer. A high frequency of K-ras mutations has been observed in the endometrium of women treated with TAM. Human DNA polymerase (pol) eta and pol kappa are highly expressed in the reproductive organs and are associated with translesion synthesis past bulky DNA adducts. To explore the miscoding properties of alpha-(N2-deoxyguanosinyl)tamoxifen (dG-N2-TAM), a major TAM-DNA adduct, site-specifically modified oligodeoxynucleotides containing a single diastereoisomer of trans or cis forms of dG-N2-TAM were prepared by phosphoramidite chemical procedure and used as templates. The primer extension reaction catalyzed by pol kappa deltaC, a truncated form of pol kappa, extended more efficiently past the adduct than that of pol eta by incorporating dCMP, a correct base, opposite the adduct. With pol eta, all diastereoisomers of dG-N2-TAM promoted small amounts of direct incorporation of dAMP and deletions. With pol kappa deltaC, dG-N2-TAM promoted small amounts of dTMP and/or dAMP incorporations and deletions. The miscoding properties varied depending on the diastereoisomer of dG-N2-TAM adducts and the DNA pol used. Steady-state kinetic studies were also performed using either the nonspecific sequence or the K-ras gene sequence containing a single dG-N2-TAM at the second base of codon 12. With pol eta, the bypass frequency past the dA x dG-N2-TAM pair positioned in the K-ras sequence was only 2.3 times lower than that for the dC x dG-N2-TAM pair, indicating that dG-N2-TAM in the K-ras sequence has higher miscoding potential than that in the nonspecific sequence. However, with pol kappa deltaC, the bypass frequency past the dC x dG-N2-TAM pair was higher than that of the dT x dG-N2-TAM pair in both sequences. The properties of pol eta and pol kappa are consistent with the mutagenic events attributed to TAM-DNA adducts.     

Misincorporation of dAMP opposite 2-hydroxyadenine, an oxidative form of adenine.

Nucleotide incorporation opposite an oxidative form of adenine, 2-hydroxyadenine (2-OH-Ade) was investigated. When a primed template with 2-OH-Ade was treated with an exonuclease-deficient Klenow fragment of Escherichia coli DNA polymerase I (KFexo-), recombinant rat DNA polymerase beta (pol beta) or calf thymus DNA polymerase alpha (pol alpha), incorporation of dTMP and dAMP was observed. In addition, KFexo- inserted dGMP as well. A steady-state kinetic study indicated that the insertion of dAMP and dTMP opposite the DNA lesion occurred with similar frequency with KFexo- and pol beta. Insertion of dTMP opposite 2-OH-Ade was favored to that of dAMP by pol alpha. Chain extension from the A.2-OH-Ade pair is less favored than that from the T.2-OH-Ade pair by all three DNA polymerase. Analysis of full-length products of in vitro DNA synthesis showed that dTMP and dAMP were incorporated by DNA polymerases and that exonuclease-proficient and -deficient Klenow fragments also inserted dGMP opposite 2-OH-Ade. These results suggest that formation of 2-OH-Ade from A in DNA will induce A-->T and A-->C transversions in cells.     

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.     

Translesion synthesis past equine estrogen-derived 2'-deoxycytidine DNA adducts by human DNA polymerases eta and kappa

Estrogen replacement therapy (ERT), composed of equilenin, is associated with increased risk of breast, ovarian, and endometrial cancers. Several diastereoisomers of unique dC and dA DNA adducts were derived from 4-hydroxyequilenin (4-OHEN), a metabolite of equilenin, and have been detected in women receiving ERT. To explore the miscoding property of 4-OHEN-dC adduct, site-specifically modified oligodeoxynucleotides (Pk-1, Pk-2, Pk-3, and Pk-4) containing a single diastereoisomer of 4-OHEN-dC were prepared by a postsynthetic method. Among them, major 4-OHEN-dC-modified oligodeoxynucleotides (Pk-3 and Pk-4) were used to prepare the templates for primer extension reactions catalyzed by DNA polymerase (pol) alpha, pol eta, and pol kappa. Primer extension was retarded one base prior to the lesion and opposite the lesion; stronger blockage was observed with pol alpha, while with human pol eta or pol kappa, a fraction of the primers was extended past the lesion. Steady-state kinetic studies showed that both pol kappa and pol eta inserted dCMP and dAMP opposite the 4-OHEN-dC and extended past the lesion. Never or less-frequently, dGMP, the correct base, was inserted opposite the lesion. The relative bypass frequency past the 4-OHEN-dC lesion with pol eta was at least 3 orders of magnitude higher than that for pol kappa, as observed for primer extension reactions. The bypass frequency past the dA.4-OHEN-dC adduct in Pk-4 was 2 orders of magnitude more efficient than that past the adduct in Pk-3. Thus, 4-OHEN-dC is a highly miscoding lesion capable of generating C --> T transitions and C --> G transversions. The miscoding frequency and specificity of 4-OHEN-dC were strikingly influenced by the adduct stereochemistry and DNA polymerase used.     

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.     

In vivo formation and in vitro replication of a guanine-thymine intrastrand cross-link lesion

G[8-5m]T, a guanine-thymine intrastrand cross-link lesion where the C8 of guanine is covalently bonded to the neighboring 3'-thymine through its methyl carbon, was previously shown to form in an aqueous solution of duplex DNA upon exposure to gamma- or X-rays and in calf thymus DNA treated with Fenton reagents. Here, we employed LC-MS/MS and demonstrated for the first time that this lesion could be induced in a dose-dependent manner in human Hela-S3 cells upon exposure to gamma-rays. We further carried out in vitro replication studies on a substrate containing a site-specifically incorporated G[8-5m]T, and our results showed that the Klenow fragment of Escherichia coli DNA polymerase I stopped synthesis mostly after incorporating the correct nucleotide dAMP opposite the 3'-thymine moiety of the lesion. On the other hand, yeast Saccharomyces cerevisiae DNA polymerase eta (pol eta) was able to replicate past the cross-link lesion, but with markedly reduced efficiency in nucleotide incorporation opposite the 5'-guanine of the lesion. Steady-state kinetic analyses for nucleotide incorporation by yeast pol eta showed that the 5'-guanine portion of the lesion also decreased pronouncedly the fidelity of nucleotide incorporation; the insertion of dAMP and dGMP was favored over that of the correct nucleotide, dCMP. The above results support the conclusion that oxidative intrastrand cross-link lesions, if not repaired, can be cytotoxic and mutagenic.     

Role of base stacking and sequence context in the inhibition of yeast DNA polymerase eta by pyrene nucleotide

The Y family DNA polymerase yeast pol eta inserts pyrene deoxyribose monophosphate (dPMP) in preference to A opposite an abasic site, the 3'-T of a thymine dimer, and a normal T with almost equal efficiency. In contrast, pol A family polymerases such as Klenow fragment and T7 DNA polymerase only insert dPMP efficiently opposite an abasic site and the 3'-T of a thymine dimer but not opposite undamaged DNA. Pyrene nucleotide is also an efficient chain-terminating inhibitor of DNA synthesis by pol eta but not by Klenow fragment or T7 DNA polymerase. To better understand the origin of the efficiency and sequence specificity of dPMP insertion by pol eta, the kinetics of dPMP insertion opposite various templates have been determined. In one sequence context, the efficiency of dPMP insertion increases 4.6-fold opposite G < A < T < C, suggesting that the templating nucleotide modulates dPMP insertion efficiency by having to destack prior to dPTP binding. The efficiency of insertion of dPMP opposite T in the same sequence context increases 7-fold for primers terminating in G < A < C < T and is similar to that observed for nontemplated blunt-end extension, suggesting that stacking interactions between the pyrene and the primer terminus are also important. On heterogeneous templates, the average selectivity for dPMP insertion relative to the complementary dNMP decreases in the order of dAMP > dGMP > dTMP > dCMP, from a high of 5.8 when dAMP is to be inserted following a T to a low of 0.5 when dCMP is to be inserted following a C. The relative preference for dPMP insertion at a given site can be largely explained by the energetic cost of destacking the templating base and stacking of pyrene nucleotide relative to that of stacking and base pairing the complementary nucleotide. Thus, pyrene nucleotide represents a novel class of nucleotide-based chain-terminating DNA synthesis inhibitors whose base portion consists of a hydrophobic, non-hydrogen bonding, base-pair mimic.     

Abasic template lesions are strong chain terminators for DNA primase but not for DNA polymerase alpha during the synthesis of new DNA strands.

The effects of abasic lesions on both primase activity and DNA polymerase alpha- (pol alpha) catalyzed elongation of primase-synthesized primers were examined. Abasic lesions were strong chain terminators during primer synthesis by primase. However, extension of primase-synthesized primers by pol alpha resulted in 60-93% bypass of abasic lesions. Sequencing of bypass products generated during this primase-coupled pol alpha activity showed that dAMP was preferentially incorporated opposite the abasic lesion, indicating that pol alpha was responsible for bypass. In contrast, previous analyses of pol alpha-catalyzed elongation of exogenously supplied DNA primer-templates showed that abasic lesions strongly terminated DNA synthesis. Thus, elongation of primase-synthesized primers by pol alpha-primase is fundamentally different than elongation of exogenously added primer-templates with respect to interaction with abasic lesions. Furthermore, this high level of abasic lesion bypass during primase-coupled pol alpha activity provides an additional mechanism for how translesional synthesis may occur in vivo, an event hypothesized to be mutagenic.     

LC-MS/MS identification and yeast polymerase eta bypass of a novel gamma-irradiation-induced intrastrand cross-link lesion G[8-5]C

Reactive oxygen species can give rise to intrastrand cross-link lesions, where two neighboring nucleobases are covalently bonded. Here, we employed LC-MS/MS and demonstrated for the first time that gamma irradiation of a synthetic duplex oligodeoxyribonucleotide can give rise to an intrastrand cross-link lesion G[8-5]C, where the C8 carbon atom of guanine and the C5 carbon atom of its 3'-neighboring cytosine are covalently bonded. We also carried out in vitro replication studies of a substrate containing a site-specifically incorporated G[8-5]C, and our results showed that yeast Saccharomyces cerevisiae DNA polymerase eta (pol eta) was able to replicate past the cross-link lesion. Steady-state kinetic analyses for nucleotide incorporation by pol eta showed that the 3'-cytosine moiety of the cross-link did not significantly affect either the efficiency or the fidelity of nucleotide incorporation. The 5' guanine portion of the cross-link lesion, however, markedly reduced both the efficiency and the fidelity of nucleotide incorporation; the insertion of dGMP or dAMP was slightly favored over the insertion of the correct nucleotide, dCMP, which was in turn favored over the insertion of dTMP. The above results support that the oxidative cross-link lesion, if not repaired, can be mutagenic.     

The Gly-952 residue of Saccharomyces cerevisiae DNA polymerase alpha is important in discriminating correct deoxyribonucleotides from incorrect ones

Gly-952 is a conserved residue in Saccharomyces cerevisiae DNA polymerase alpha (pol alpha) that is strictly required for catalytic activity and for genetic complementation of a pol alpha-deficient yeast strain. This study analyzes the role of Gly-952 by characterizing the biochemical properties of Gly-952 mutants. Analysis of the nucleotide incorporation specificity of pol alpha G952A showed that this mutant incorporates nucleotides with extraordinarily low fidelity. In a steady-state kinetic assay to measure nucleotide misincorporation, pol alpha G952A incorporated incorrect nucleotides more efficiently than correct nucleotides opposite template C, G, and T. The fidelity of the G952A mutant polymerase was highest at template A, where the ratio of incorporation of dCMP to dTMP was as high as 0.37. Correct nucleotide insertion was 500- to 3500-fold lower for G952A than for wild type pol alpha, with up to 22-fold increase in pyrimidine misincorporation. The Km for G952A pol alpha bound to mismatched termini T:T, T:C, C:A, and A:C was 71- to 460-fold lower than to a matched terminus. Furthermore, pol alpha G952A preferentially incorporated pyrimidine instead of dAMP opposite an abasic site, cis-syn cyclobutane di-thymine, or (6-4) di-thymine photoproduct. These data demonstrate that Gly-952 is a critical residue for catalytic efficiency and error prevention in S. cerevisiae pol alpha.     

Mutational specificity of gamma-radiation-induced guanine-thymine and thymine-guanine intrastrand cross-links in mammalian cells and translesion synthesis past the guanine-thymine lesion by human DNA polymerase eta

Comparative mutagenesis of gamma- or X-ray-induced tandem DNA lesions G[8,5-Me]T and T[5-Me,8]G intrastrand cross-links was investigated in simian (COS-7) and human embryonic (293T) kidney cells. For G[8,5-Me]T in 293T cells, 5.8% of progeny contained targeted base substitutions, whereas 10.0% showed semitargeted single-base substitutions. Of the targeted mutations, the G --> T mutation occurred with the highest frequency. The semitargeted mutations were detected up to two bases 5' and three bases 3' to the cross-link. The most prevalent semitargeted mutation was a C --> T transition immediately 5' to the G[8,5-Me]T cross-link. Frameshifts (4.6%) (mostly small deletions) and multiple-base substitutions (2.7%) also were detected. For the T[5-Me,8]G cross-link, a similar pattern of mutations was noted, but the mutational frequency was significantly higher than that of G[8,5-Me]T. Both targeted and semitargeted mutations occurred with a frequency of approximately 16%, and both included a dominant G --> T transversion. As in 293T cells, more than twice as many targeted mutations in COS cells occurred in T[5-Me,8]G (11.4%) as in G[8,5-Me]T (4.7%). Also, the level of semitargeted single-base substitutions 5' to the lesion was increased and 3' to the lesion decreased in T[5-Me,8]G relative to G[8,5-Me]T in COS cells. It appeared that the majority of the base substitutions at or near the cross-links resulted from incorporation of dAMP opposite the template base, in agreement with the so-called "A-rule". To determine if human polymerase eta (hpol eta) might be involved in the mutagenic bypass, an in vitro bypass study of G[8,5-Me]T in the same sequence was carried out, which showed that hpol eta can bypass the cross-link incorporating the correct dNMP opposite each cross-linked base. For G[8,5-Me]T, nucleotide incorporation by hpol eta was significantly different from that by yeast pol eta in that the latter was more error-prone opposite the cross-linked Gua. The incorporation of the correct nucleotide, dAMP, by hpol eta opposite cross-linked T was 3-5-fold more efficient than that of a wrong nucleotide, whereas incorporation of dCMP opposite the cross-linked G was 10-fold more efficient than that with dTMP. Therefore, the nucleotide incorporation pattern by hpol eta was not consistent with the observed cellular mutations. Nevertheless, at and near the lesion, hpol eta was more error-prone compared to a control template. The in vitro data suggest that translesion synthesis by another Y-family DNA polymerase and/or flawed participation of an accessory protein is a more likely scenario in the mutagenesis of these lesions in mammalian cells. However, hpol eta may play a role in correct bypass of the cross-links.     

Yeast pol eta holds a cis-syn thymine dimer loosely in the active site during elongation opposite the 3'-T of the dimer,but tightly opposite the 5'-T

Polymerase eta is a member of the Y family of DNA polymerases which is able to bypass thymine dimers efficiently and in a relatively error-free manner. To elucidate the mechanism of dimer bypass, the efficiency of dAMP and pyrene nucleotide insertion opposite the thymine dimer and its N3-methyl derivatives was determined. Pol eta inserts pyrene nucleotide with greater efficiency than dAMP opposite the 3'-T of an undimerized or dimerized T and is an effective inhibitor of DNA synthesis by pol eta. Substitution of the N3H of the 3'-T of an undimerized T or a dimerized T with a methyl group has little effect on the insertion efficiency of pyrene nucleotide but greatly inhibits the insertion of dAMP. Together, these results suggest that the error-free insertion of dAMP opposite the 3'-T of the cis-syn thymine dimer happens by way of a loosely held dimer in the active site which can be displaced from the active site by pyrene nucleotide. In contrast, pol eta cannot insert pyrene nucleotide opposite the 5'-T of the dimer, whereas it can insert dAMP with efficiency comparable to that opposite the 3'-T. The inability to insert pyrene nucleotide opposite the 5'-T of the dimer is consistent with the idea that while the polymerase binds loosely to a templating nucleotide, it binds tightly to the nucleotide to its 3'-side. Overall, the results show a marked difference from similar studies on pol I family polymerases, and suggest mechanisms by which this Y family polymerase can process damaged DNA efficiently.     

Miscoding events during DNA synthesis past the nitration-damaged base 8-nitroguanine

8-Nitro-2'-deoxyguanosine (8-NO(2)-dG) DNA adducts are induced by the reactive nitrogen species and may be associated with the development of cancer in inflammatory tissues. To explore the miscoding potential of 8-NO(2)-dG adduct, an oligodeoxynucleotide containing a single 8-NO(2)-dG adduct was prepared by photochemical synthesis and used as a template in primer extension reactions catalyzed by mammalian DNA polymerases (pol). Primer extension reactions catalyzed by pol alpha or beta were strongly retarded at the 8-NO(2)-dG lesion; a fraction of primers was extended past the lesion by incorporating preferentially dCMP, the correct base, opposite the lesion, accompanied by lesser amounts of dAMP and dGMP incorporation. In contrast, primer extension reactions catalyzed by pol eta or a truncated form of pol kappa (pol kappaDeltaC) readily extended past the 8-NO(2)-dG lesion. Pol eta and kappaDeltaC showed more broad miscoding spectra; direct incorporations of dCMP and dAMP were observed, along with lesser amounts of dGMP and dTMP incorporations and deletions. The miscoding frequencies induced by pol eta and kappaDeltaC were at least 8 times higher than that of pol alpha or beta. Miscoding frequency and specificity of 8-NO(2)-dG varied depending on the DNA polymerases used. These observations were supported by steady-state kinetic studies. 8-NO(2)-dG adduct may play an important role in initiating inflammation driven carcinogenesis.     

Translesion DNA synthesis by yeast DNA polymerase eta on templates containing N2-guanine adducts of 1,3-butadiene metabolites

Yeast DNA polymerase eta can replicate through cis-syn cyclobutane pyrimidine dimers and 8-oxoguanine lesions with the same efficiency and accuracy as replication of an undamaged template. Previously, it has been shown that Escherichia coli DNA polymerases I, II, and III are incapable of bypassing DNA substrates containing N(2)-guanine adducts of stereoisomeric 1,3-butadiene metabolites. Here we showed that yeast polymerase eta replicates DNA containing the monoadducts (S)-butadiene monoepoxide and (S,S)-butadiene diolepoxide N(2)-guanines albeit at an approximately 200-300-fold lower efficiency relative to the control guanine. Interestingly, nucleotide incorporation opposite the (R)-butadiene monoepoxide and the (R,R)-butadiene diolepoxide N(2)-guanines was approximately 10-fold less efficient than incorporation opposite their S stereoisomers. Polymerase eta preferentially incorporates the correct nucleotide opposite and downstream of all four adducts, except that it shows high misincorporation frequencies for elongation of C paired with (R)-butadiene monoepoxide N(2)-guanine. Additionally, polymerase eta does not bypass the (R,R)- and (S,S)-butadiene diolepoxide N(2)-guanine-N(2)-guanine intra- strand cross-links, and replication is completely blocked just prior to the lesion. Collectively, these data suggest that polymerase eta can tolerate the geometric distortions in DNA conferred by the N(2)-guanine butadiene monoadducts but not the intrastrand cross-links.     

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.