Miscoding properties of 6alpha- and 6beta-diastereoisomers of the N(2)-(estradiol-6-yl)-2'-deoxyguanosine DNA adduct by Y-family human DNA polymerases

Treatment with estrogen increases the risk of breast, ovary, and endometrial cancers in women. DNA damage induced by estrogen is thought to be involved in estrogen carcinogenesis. In fact, Y-family human DNA polymerases (pol) eta and kappa, which are highly expressed in the reproductive organs, miscode model estrogen-derived DNA adducts during DNA synthesis. Since the estrogen-DNA adducts are a mixture of 6alpha- and 6beta-diastereoisomers of dG-N(2)-6-estrogen or dA-N(6)-6-estrogen, the stereochemistry of each isomeric adduct on translesion synthesis catalyzed by DNA pols has not been investigated. We have recently established a phosphoramidite chemical procedure to insert 6alpha- or 6beta-isomeric N(2)-(estradiol-6-yl)-2'-deoxyguanosine (dG-N(2)-6-E(2)) into oligodeoxynucleotides. Using such site-specific modified oligomer as a template, the specificity and frequency of miscoding by dG-N(2)-6alpha-E(2) or dG-N(2)-6beta-E(2) were explored using pol eta and a truncated form of pol kappa (pol kappaDeltaC). Translesion synthesis catalyzed by pol eta bypassed both the 6alpha- and 6beta-isomers of dG-N(2)-6-E(2), with a weak blockage at the adduct site, while translesion synthesis catalyzed by pol kappaDeltaC readily bypassed both isomeric adducts. Quantitative analysis of base substitutions and deletions occurring at the adduct site showed that pol kappaDeltaC was more efficient than pol eta by incorporating dCMP opposite both 6alpha- and 6beta-isomeric dG-N(2)-6-E(2) adducts. The miscoding events occurred more frequently with pol eta, but not with pol kappaDeltaC. Pol eta promoted incorporation of dAMP and dTMP at both the 6alpha- and 6beta-isomeric adducts, generating G --> T transversions and G --> A transitions. One- and two-base deletions were also formed. The 6alpha-isomeric adduct promoted slightly lower frequency of dCMP incorporation and higher frequency of dTMP incorporation and one-base deletions, compared with the 6beta-isomeric adduct. These observations were supported by steady-state kinetic studies. Taken together, the miscoding property of the 6alpha-isomeric dG-N(2)-6-E(2) is likely to be similar to that of the 6beta-isomeric adduct.     

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.     

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.     

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.     

Efficient translesion replication past oxaliplatin and cisplatin GpG adducts by human DNA polymerase eta

Platinum anticancer agents form bulky DNA adducts which are thought to exert their cytotoxic effect by blocking DNA replication. Translesion synthesis, one of the pathways of postreplication repair, is thought to account for some resistance to DNA damage and much of the mutagenicity of bulky DNA adducts in dividing cells. Oxaliplatin has been shown to be effective in cisplatin-resistant cell lines and less mutagenic than cisplatin in the Ames assay. We have shown that the eukaryotic DNA polymerases yeast pol zeta, human pol beta, and human pol gamma bypass oxaliplatin-GG adducts more efficiently than cisplatin-GG adducts. Human pol eta, a product of the XPV gene, has been shown to catalyze efficient translesion synthesis past cis, syn-cyclobutane pyrimidine dimers. In the present study we compared translesion synthesis past different Pt-GG adducts by human pol eta. Our data show that, similar to other eukaryotic DNA polymerases, pol eta bypasses oxaliplatin-GG adducts more efficiently than cisplatin-GG adducts. However, pol eta-catalyzed translesion replication past Pt-DNA adducts was more efficient and less accurate than that seen for previously tested polymerases. We show that the efficiency and fidelity of translesion replication past Pt-DNA adducts appear to be determined by both the structure of the adduct and the DNA polymerase active site.     

Translesion synthesis by human DNA polymerase kappa on a DNA template containing a single stereoisomer of dG-(+)- or dG-(-)-anti-N(2)-BPDE (7,8-dihydroxy-anti-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene)

Several recently discovered human DNA polymerases are associated with translesion synthesis past DNA adducts. These include human DNA polymerase kappa (pol kappa), a homologue of Escherichia coli pol IV, which enhances the frequency of spontaneous mutation. Using a truncated form of pol kappa (pol kappa Delta C), translesion synthesis past dG-(+)- or dG-(-)-anti-N(2)-BPDE (7,8-dihydroxy-anti-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene) adducts was explored. Site-specifically-modified oligodeoxynucleotides containing a single stereoisomeric dG-N(2)-BPDE lesion were used as DNA templates for primer extension reactions catalyzed by pol kappa Delta C. Primer extension was retarded one base prior to the dG-N(2)-BPDE lesion; when incubated for longer times or with higher concentration of enzyme, full primer extension was observed. Quantitative analysis of fully extended products showed preferential incorporation of dCMP, the correct base, opposite all four stereoisomeric dG-N(2)-BPDE lesions. (+)-trans-dG-N(2)-BPDE, a major BPDE-DNA adduct, promoted small amounts of dTMP, dAMP, and dGMP misincorporation opposite the lesion (total 2.7% of the starting primers) and deletions (1.1%). Although (+)-cis-dG-N(2)-BPDE was most effective in blocking translesion synthesis, its miscoding properties were similar to other dG-N(2)-BPDE isomers. Steady-state kinetic data indicate that dCMP is efficiently inserted opposite all dG-N(2)-BPDE adducts and extended past these lesions. The relative frequency of translesion synthesis (F(ins) x F(ext)) of dC.dG-N(2)-BPDE pairs was 2-6 orders of magnitude higher than that of other mismatched pairs. Pol kappa may play an important role in translesion synthesis by incorporating preferentially the correct base opposite dG-N(2)-BPDE. Its relatively low contribution to mutagenicity suggests that other newly discovered DNA polymerase(s) may be involved in mutagenic events attributed to dG-N(2)-BPDE adducts in human cells.     

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

Newly discovered human DNA polymerase (pol) eta and kappa are highly expressed in the reproductive organs, such as testis, ovary, and uterus, where steroid hormones are produced. Because treatment with estrogen increases the risk of developing breast, ovary, and endometrial cancers, miscoding events occurring at model estrogen-derived DNA adducts were explored using pol eta and a truncated form of human pol kappa (pol kappaDeltaC). These enzymes bypassed N(2)-[3-methoxyestra-1,3,5(10)-trien-6-yl]-2'-deoxyguanosine (dG-N(2)-3MeE) and N(6)-[3-methoxyestra-1,3,5(10)-trien-6-yl]-2'-deoxyadenosine (dA-N(6)-3MeE), which were embedded in site-specifically modified oligodeoxynucleotide templates. Quantitative analysis of base substitutions and deletions occurring at the lesion site showed that pol kappaDeltaC was more efficient at incorporating dCMP opposite the dG-N(2)-3MeE lesion than pol eta. Surprisingly, the frequency of translesion synthesis beyond the dC*dG-N(2)-3MeE pair was 13% of the normal dC*dG pair and was 4 and 6 orders of magnitude higher than that of dC*(+)-trans-dG-N(2)-benzo[a]pyrene and dC*dG-C8-acetylaminofluorene pairs, respectively, suggesting that dG-N(2)-3MeE is a natural substrate for pol kappa. In contrast, the bypass frequency beyond the dT*dA-N(6)-3MeE pair was 7 orders of magnitude less than that for the normal dT*dA pair. dA-N(6)-3MeE is a more miscoding lesion than dG-N(2)-3MeE. Pol eta promoted incorporation of dAMP and dCMP at the dA-N(6)-3MeE lesion, while with pol kappaDeltaC, deletions were more frequently observed, along with incorporation of dAMP and dCMP opposite the lesion. These observations were also supported by steady-state kinetic studies. When taken together, the properties of pol eta and kappa are consistent with the mutagenic events attributed to estrogen-derived DNA adducts.     

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.     

Mammalian translesion DNA synthesis across an acrolein-derived deoxyguanosine adduct. Participation of DNA polymerase eta in error-prone synthesis in human cells

alpha-OH-PdG, an acrolein-derived deoxyguanosine adduct, inhibits DNA synthesis and miscodes significantly in human cells. To probe the cellular mechanism underlying the error-free and error-prone translesion DNA syntheses, in vitro primer extension experiments using purified DNA polymerases and site-specific alpha-OH-PdG were conducted. The results suggest the involvement of pol eta in the cellular error-prone translesion synthesis. Experiments with xeroderma pigmentosum variant cells, which lack pol eta, confirmed this hypothesis. The in vitro results also suggested the involvement of pol iota and/or REV1 in inserting correct dCMP opposite alpha-OH-PdG during error-free synthesis. However, none of translesion-specialized DNA polymerases catalyzed significant extension from a dC terminus when paired opposite alpha-OH-PdG. Thus, our results indicate the following. (i) Multiple DNA polymerases are involved in the bypass of alpha-OH-PdG in human cells. (ii) The accurate and inaccurate syntheses are catalyzed by different polymerases. (iii) A modification of the current eukaryotic bypass model is necessary to account for the accurate bypass synthesis in human cells.     

Translesion synthesis past acrolein-derived DNA adduct,gamma -hydroxypropanodeoxyguanosine,by yeast and human DNA polymerase eta

gamma-Hydroxy-1,N(2)-propano-2'deoxyguanosine (gamma-HOPdG) is a major deoxyguanosine adduct derived from acrolein, a known mutagen. In vitro, this adduct has previously been shown to pose a severe block to translesion synthesis by a number of polymerases (pol). Here we show that both yeast and human pol eta can incorporate a C opposite gamma-HOPdG at approximately 190- and approximately 100-fold lower efficiency relative to the control deoxyguanosine and extend from a C paired with the adduct at approximately 8- and approximately 19-fold lower efficiency. Although DNA synthesis past gamma-HOPdG by yeast pol eta was relatively accurate, the human enzyme misincorporated nucleotides opposite the lesion with frequencies of approximately 10(-1) to 10(-2). Because gamma-HOPdG can adopt both ring closed and ring opened conformations, comparative replicative bypass studies were also performed with two model adducts, propanodeoxyguanosine and reduced gamma-HOPdG. For both yeast and human pol eta, the ring open reduced gamma-HOPdG adduct was less blocking than gamma-HOPdG, whereas the ring closed propanodeoxyguanosine adduct was a very strong block. Replication of DNAs containing gamma-HOPdG in wild type and xeroderma pigmentosum variant cells revealed a somewhat decreased mutation frequency in xeroderma pigmentosum variant cells. Collectively, the data suggest that pol eta might potentially contribute to both error-free and mutagenic bypass of gamma-HOPdG.     

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.     

The Y-family DNA polymerase kappa (pol kappa) functions in mammalian nucleotide-excision repair

DNA polymerase kappa (pol kappa) is a member of the Y-family of DNA polymerases that are thought to function in translesion synthesis (TLS) past different types of DNA damage. Here, we show that pol kappa-deficient mouse cells have substantially reduced (but not absent) levels of nucleotide excision repair (NER) of UV damage, as measured by several methods. Our results provide evidence for an unexpected role for pol kappa in mammalian NER.     

Error prone translesion synthesis past gamma-hydroxypropano deoxyguanosine, the primary acrolein-derived adduct in mammalian cells

8-Hydroxy-5,6,7,8-tetrahydropyrimido[1,2-a]purin- 10(3H)-one,3-(2'-deoxyriboside) (1,N(2)-gamma-hydroxypropano deoxyguanosine, gamma-HOPdG) is a major DNA adduct that forms as a result of exposure to acrolein, an environmental pollutant and a product of endogenous lipid peroxidation. gamma-HOPdG has been shown previously not to be a miscoding lesion when replicated in Escherichia coli. In contrast to those prokaryotic studies, in vivo replication and mutagenesis assays in COS-7 cells using single stranded DNA containing a specific gamma-HOPdG adduct, revealed that the gamma-HOPdG adduct was significantly mutagenic. Analyses revealed both transversion and transition types of mutations at an overall mutagenic frequency of 7.4 x 10(-2)/translesion synthesis. In vitro gamma-HOPdG strongly blocks DNA synthesis by two major polymerases, pol delta and pol epsilon. Replicative blockage of pol delta by gamma-HOPdG could be diminished by the addition of proliferating cell nuclear antigen, leading to highly mutagenic translesion bypass across this adduct. The differential functioning and processing capacities of the mammalian polymerases may be responsible for the higher mutation frequencies observed in this study when compared with the accurate and efficient nonmutagenic bypass observed in the bacterial system.     

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.     

8-(Hydroxymethyl)-3,N(4)-etheno-C, a potential carcinogenic glycidaldehyde product, miscodes in vitro using mammalian DNA polymerases

8-(Hydroxymethyl)-3,N(4)-etheno-C (8-HM-epsilonC) is an exocyclic adduct resulting from the reaction of dC with glycidaldehyde, a mutagen and animal carcinogen. This compound has now been synthesized and its phosphoramidite incorporated site-specifically into a defined 25-mer oligonucleotide. In this study, the mutagenic potential of this adduct in the 25-mer oligonucleotide was investigated in an in vitro primer-template extension assay using four mammalian DNA polymerases. The miscoding potentials were also compared to those of an analogous derivative, 3,N(4)-etheno C (epsilonC), in the same sequence. Both adducts primarily blocked replication by calf thymus DNA polymerase alpha at the modified base, while human polymerase beta catalyzed measurable replication synthesis through both adducts. Nucleotide insertion experiments showed that dA and dC were incorporated by pol beta opposite either adduct, which would result in a C --> T transition or C --> G transversion. Human polymerase eta, a product of the xeroderma pigmentosum variant (XP-V) gene, catalyzed the most efficient bypass of the two lesions with 25% and 32% for 8-HM-epsilonC and epsilonC bypassed after 15 min. Varying amounts of all four bases opposite the modified bases resulted with pol eta. Human polymerase kappa primarily blocked synthesis at the base prior to the adduct. However, some specific misincorporation of dT resulted, forming an epsilonC.T or 8-HM-epsilonC.T pair. From these data, we conclude that the newly synthesized glycidaldehyde-derived adduct, 8-HM-epsilonC, is a miscoding lesion. The bypass efficiency and insertion specificity of 8-HM-epsilonC and epsilonC were similar for all four polymerases tested, which could be attributed to the similar planarity and sugar conformations for these two derivatives as demonstrated by molecular modeling studies.     

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.     

Frameshifts and deletions during in vitro translesion synthesis past Pt-DNA adducts by DNA polymerases beta and eta

DNA polymerases beta (pol beta ) and eta (pol eta ) are the only two eukaryotic polymerases known to efficiently bypass cisplatin and oxaliplatin adducts in vitro. Frameshift errors are an important aspect of mutagenesis. We have compared the types of frameshifts that occur during translesion synthesis past cisplatin and oxaliplatin adducts in vitro by pol beta and pol eta on a template containing multiple runs of nucleotides flanking a single platinum-GG adduct. Translesion synthesis past platinum adducts by pol beta resulted in approximately 50% replication products containing single-base deletions. For both adducts the majority of -1 frameshifts occurred in a TTT sequence 3-5 bp upstream of the DNA lesion. For pol eta, all of the bypass products for both cisplatin and oxaliplatin adducts contained -1 frameshifts in the upstream TTT sequence and most of the products of replication on oxaliplatin-damaged templates had multiple replication errors, both frameshifts and misinsertions. In addition, on platinated templates both polymerases generated replication products 4-8 bp shorter than the full-length products. The majority of short cisplatin-induced products contained an internal deletion which included the adduct. In contrast, the majority of oxaliplatin-induced short products contained a 3' terminal deletion. The implications of these in vitro results for in vivo mutagenesis are discussed.     

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 across bulky N2-alkyl guanine DNA adducts by human DNA polymerase kappa

DNA polymerase (pol) kappa is one of the so-called translesion polymerases involved in replication past DNA lesions. Bypass events have been studied with a number of chemical modifications with human pol kappa, and the conclusion has been presented, based on limited quantitative data, that the enzyme is ineffective at incorporating opposite DNA damage but proficient at extending beyond bases paired with the damage. Purified recombinant full-length human pol kappa was studied with a series of eight N(2)-guanyl adducts (in oligonucleotides) ranging in size from methyl- to -CH(2)(6-benzo[a]pyrenyl) (BP). Steady-state kinetic parameters (catalytic specificity, k(cat)/K(m)) were similar for insertion of dCTP opposite the lesions and for extension beyond the N(2)-adduct G:C pairs. Mispairing of dGTP and dTTP was similar and occurred with k(cat)/K(m) values approximately 10(-3) less than for dCTP with all adducts; a similar differential was found for extension beyond a paired adduct. Pre-steady-state kinetic analysis showed moderately rapid burst kinetics for dCTP incorporations, even opposite the bulky methyl(9-anthracenyl)- and BPG adducts (k(p) 5.9-10.3 s(-1)). The rapid bursts were abolished opposite BPG when alpha-thio-dCTP was used instead of dCTP, implying rate-limiting phosphodiester bond formation. Comparisons are made with similar studies done with human pols eta and iota; pol kappa is the most resistant to N(2)-bulk and the most quantitatively efficient of these in catalyzing dCTP incorporation opposite bulky guanine N(2)-adducts, particularly the largest (N(2)-BPG).     

Translesion synthesis across O6-alkylguanine DNA adducts by recombinant human DNA polymerases

Previous studies have shown that replicative bacterial and viral DNA polymerases are able to bypass the mutagenic lesions O(6)-methyl and -benzyl (Bz) G. Recombinant human polymerase (pol) delta also copied past these two lesions but was totally blocked by O(6)-[4-oxo-4-(3-pyridyl)butyl] (Pob)G, an important mutagenic lesion formed following metabolic activation of the tobacco-specific carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone. The human translesion pols iota and kappa produced mainly only 1-base incorporation opposite O(6)-MeG and O(6)-BzG and had very low activity in copying O(6)-PobG. Human pol eta copied past all three adducts. Steady-state kinetic analysis showed similar efficiencies of insertion opposite the O(6)-alkylG adducts for dCTP and dTTP with pol eta and kappa; pol iota showed a strong preference for dTTP. pol eta, iota, and kappa showed pre-steady-state kinetic bursts for dCTP incorporation opposite G and O(6)-MeG but little, if any, for O(6)-BzG or O(6)-PobG. Analysis of the pol eta O(6)-PobG products indicated that the insertion of G was opposite the base (C) 5' of the adduct, but this product was not extended. Mass spectrometry analysis of all of the pol eta primer extension products indicated multiple components, mainly with C or T inserted opposite O(6)-alkylG but with no deletions in the cases of O(6)-MeG and O(6)-PobG. With pol eta and O(6)-BzG, products were also obtained with -1 and -2 deletions and also with A inserted (opposite O(6)-BzG). The results with pol eta may be relevant to some mutations previously reported with O(6)-alkylG adducts in mammalian cells.