Role of DNA polymerase eta in the UV mutation spectrum in human cells

In humans, inactivation of the DNA polymerase eta gene (pol eta) results in sunlight sensitivity and causes the cancer-prone xeroderma pigmentosum variant syndrome (XP-V). Cells from XP-V individuals have a reduced capacity to replicate UV-damaged DNA and show hypermutability after UV exposure. Biochemical assays have demonstrated the ability of pol eta to bypass cis-syn-cyclobutane thymine dimers, the most common lesion generated in DNA by UV. In most cases, this bypass is error-free. To determine the actual requirement of pol eta in vivo, XP-V cells (XP30RO) were complemented by the wild type pol eta gene. We have used two pol eta-corrected clones to study the in vivo characteristics of mutations produced by DNA polymerases during DNA synthesis of UV-irradiated shuttle vectors transfected into human host cells, which had or had not been exposed previously to UV radiation. The functional complementation of XP-V cells by pol eta reduced the mutation frequencies both at CG and TA base pairs and restored UV mutagenesis to a normal level. UV irradiation of host cells prior to transfection strongly increased the mutation frequency in undamaged vectors and, in addition, especially in the pol eta-deficient XP30RO cells at 5'-TT sites in UV-irradiated plasmids. These results clearly show the protective role of pol eta against UV-induced lesions and the activation by UV of pol eta-independent mutagenic processes.     

Sequence context-dependent replication of DNA templates containing UV-induced lesions by human DNA polymerase iota

Humans possess four Y-family polymerases: pols eta, iota, kappa and the Rev1 protein. The pivotal role that pol eta plays in protecting us from UV-induced skin cancers is unquestioned given that mutations in the POLH gene (encoding pol eta), lead to the sunlight-sensitive and cancer-prone xeroderma pigmentosum variant phenotype. The roles that pols iota, kappa and Rev1 play in the tolerance of UV-induced DNA damage is, however, much less clear. For example, in vitro studies in which the ability of pol iota to bypass UV-induced cyclobutane pyrimidine dimers (CPDs) or 6-4 pyrimidine-pyrimidone (6-4PP) lesions has been assayed, are somewhat varied with results ranging from limited misinsertion opposite CPDs to complete lesion bypass. We have tested the hypothesis that such discrepancies might have arisen from different assay conditions and local sequence contexts surrounding each UV-photoproduct and find that pol iota can facilitate significant levels of unassisted highly error-prone bypass of a T-T CPD, particularly when the lesion is located in a 3'-A[T-T]A-5' template sequence context and the reaction buffer contains no KCl. When encountering a T-T 6-4PP dimer under the same assay conditions, pol iota efficiently and accurately inserts the correct base, A, opposite the 3'T of the 6-4PP by factors of approximately 10(2) over the incorporation of incorrect nucleotides, while incorporation opposite the 5'T is highly mutagenic. Pol kappa has been proposed to function in the bypass of UV-induced lesions by helping extend primers terminated opposite CPDs. However, we find no evidence that the combined actions of pol iota and pol kappa result in a significant increase in bypass of T-T CPDs when compared to pol iota alone. Our data suggest that under certain conditions and sequence contexts, pol iota can bypass T-T CPDs unassisted and can efficiently incorporate one or more bases opposite a T-T 6-4PP. Such biochemical activities may, therefore, be of biological significance especially in XP-V cells lacking the primary T-T CPD bypassing enzyme, pol eta.     

Translesion synthesis by human DNA polymerase eta across thymine glycol lesions

The XP-V (xeroderma pigmentosum variant) gene product, human DNA polymerase eta (pol eta), catalyzes efficient and accurate translesion synthesis (TLS) past cis-syn thymine-thymine dimers (TT dimer). In addition, recent reports suggest that pol eta is involved in TLS past various other types of lesion, including an oxidative DNA damage, 8-hydroxyguanine. Here, we compare the abilities of pol alpha and pol eta to replicate across thymine glycol (Tg) using purified synthetic oligomers containing a 5R- or 5S-Tg. DNA synthesis by pol alpha was inhibited at both steps of insertion of a nucleotide opposite the lesion and extension from the resulting product, indicating that pol alpha can weakly contribute to TLS past Tg lesions. In contrast, pol eta catalyzed insertion opposite the lesion as efficient as that opposite undamaged T, while extension was inhibited especially on the 5S-Tg template. Thus, pol eta catalyzed relatively efficient TLS past 5R-Tg than 5S-Tg. To compare the TLS abilities of pol eta for these lesions, we determined the kinetic parameters of pol eta for catalyzing TLS past a TT dimer, an N-2-acetylaminofluorene-modified guanine, and an abasic site analogue. The possible mechanisms of pol eta-catalyzed TLS are discussed on the basis of these results.     

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.     

DNA polymerase iota-dependent translesion replication of uracil containing cyclobutane pyrimidine dimers

Analysis of the spectrum of UV-induced mutations generated in synchronized wild-type S-phase cells reveals that only approximately 25% of mutations occur at thymine (T), whilst 75% are targeted to cytosine (C). The mutational spectra changes dramatically in XP-V cells, devoid of poleta, where approximately 45% of mutations occur at Ts and approximately 55% at Cs. At the present time, it is unclear whether the C-->T mutations actually represent true misincorporations opposite C, or perhaps occur as the result of the correct incorporation of adenine (A) opposite a C in a UV-photoproduct that had undergone deamination to uracil (U). In order to assess the role that human poliota might play, if any, in the replicative bypass of such UV-photoproducts, we have analyzed the efficiency and fidelity of pol iota-dependent bypass of a T-U cyclobutane pyrimidine dimer (CPD) in vitro. Interestingly, pol iota-dependent bypass of a T-U CPD occurs more efficiently than that of a corresponding T-T CPD. Guanine (G) was misincorporated opposite the 3'U of the T-U CPD only two-fold less frequently than the correct Watson-Crick base, A. While pol iota generally extended the G:3'U-CPD mispairs less efficiently than the correctly paired primer, pol iota-dependent extension was equal to, or greater than that observed with human pols eta and kappa and S. cerevisiae pol zeta under the same assay conditions. Thus, we hypothesize that the ability of pol iota to bypass T-U CPDs through the frequent misincorporation of G opposite the 3'U of the CPD, may provide a mechanism whereby human cells can decrease the mutagenic potential of these lesions.     

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.     

Inefficient bypass of an abasic site by DNA polymerase eta

DNA polymerase eta (Pol eta) bypasses a cis-syn thymine-thymine dimer efficiently and accurately, and inactivation of Pol eta in humans results in the cancer-prone syndrome, the variant form of xeroderma pigmentosum. Also, Pol eta bypasses the 8-oxoguanine lesion efficiently by predominantly inserting a C opposite this lesion, and it bypasses the O(6)-methylguanine lesion by inserting a C or a T. To further assess the range of DNA lesions tolerated by Pol eta, here we examine the bypass of an abasic site, a prototypical noninstructional lesion. Steady-state kinetic analyses show that both yeast and human Pol eta are very inefficient in both inserting a nucleotide opposite an abasic site and in extending from the nucleotide inserted. Hence, Pol eta bypasses this lesion extremely poorly. These results suggest that Pol eta requires the presence of template bases opposite both the incoming nucleotide and the primer terminus to catalyze efficient nucleotide incorporation.     

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.     

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.     

Human DNA polymerase lambda functionally and physically interacts with proliferating cell nuclear antigen in normal and translesion DNA synthesis

Proliferating cell nuclear antigen (PCNA) has been shown to interact with a variety of DNA polymerases (pol) such as pol delta, pol epsilon, pol iota, pol kappa, pol eta, and pol beta. Here we show that PCNA directly interacts with the newly discovered pol lambda cloned from human cells. This interaction stabilizes the binding of pol lambda to the primer template, thus increasing its affinity for the hydroxyl primer and its processivity in DNA synthesis. However, no effect of PCNA was detected on the rate of nucleotide incorporation or discrimination efficiency by pol lambda. PCNA was found to stimulate efficient synthesis by pol lambda across an abasic (AP) site. When compared with pol delta, human pol lambda showed the ability to incorporate a nucleotide in front of the lesion. Addition of PCNA led to efficient elongation past the AP site by pol lambda but not by pol delta. However, when tested on a template containing a bulky DNA lesion, such as the major cisplatin Pt-d(GpG) adduct, PCNA could not allow translesion synthesis by pol lambda. Our results suggest that the complex between PCNA and pol lambda may play an important role in the bypass of abasic sites in human cells.     

Insertion of dGMP and dAMP during in vitro DNA synthesis opposite an oxidized form of 7,8-dihydro-8-oxoguanine.

Oxidative damage to DNA bases commonly resultsin the formation of oxidized purines, particularly 7,8-dihydro-8-oxoguanine (8-oxoG) and 7,8-dihydro-8-oxoadenine (8-oxoA), the former being a well-known mutagenic lesion. Since 8-oxoG is readily subject to further oxidation compared with normal bases, the insertion of a base during DNA synthesis opposite an oxidized form of 8-oxoG was investigated in vitro. A synthetic template containing a single 8-oxoG lesion was first treated with different one-electron oxidants or under singlet oxygen conditions and then subjected to primer extension catalyzed by Klenow fragment exo- (Kf exo-), calf thymus DNA polymerase alpha (pol alpha) or human DNA polymerase beta (pol beta). Consistent with previous reports, dAMP and dCMP are inserted selectively opposite 8-oxoG with all three DNA polymerases. Interestingly, oxidation of 8-oxoG was found to induce dAMP and dGMP insertion opposite the lesion by Kf exo- with transient inhibition of primer extension occurring at the site of the modified base. Furthermore, the lesion constitutes a block during DNA synthesis by pol alpha and pol beta. Experiments with an 8-oxoA-modified template oligonucleotide show that both 8-oxoA and an oxidized form of 8-oxoA direct insertion of dTMP by Kf exo-. Mass spectrometric analysis of 8-oxoG-containing oligonucleotides before and after oxidation with IrCl62-are consistent with oxidation of primarily the 8-oxoG site, resulting in formation of a guanidinohydantoin moiety as the major product. No evidence for formation of abasic sites was obtained. These results demonstrate that an oxidized form of 8-oxoG, possibly guanidinohydantoin, may direct misreading and misinsertion of dNTPs during DNA synthesis. If such a process occurred in vivo, it would represent a point mutagenic lesion leading to G-->T and G-->C transversions. However, the corresponding oxidized form of 8-oxoA primarily shows correct insertion of T during DNA synthesis with Kf exo-.     

Adduct size limits efficient and error-free bypass across bulky N2-guanine DNA lesions by human DNA polymerase eta

The N2 position of guanine (G) is one of the major sites for DNA modification by various carcinogens. Eight oligonucleotides with varying adduct bulk at guanine N2 were analyzed for catalytic efficiency and fidelity with human DNA polymerase (pol) eta, which is involved in translesion synthesis (TLS). Pol eta effectively bypassed N2-methyl(Me)G, N2-ethyl(Et)G, N2-isobutyl(Ib)G, N2-benzyl(Bz)G, and N2-CH2(2-naphthyl)G but was severely blocked at N2-CH2(9-anthracenyl)G (N2-AnthG) and N2-CH2(6-benzo[a]pyrenyl)G (N2-BPG). Steady-state kinetic analysis showed proportional decreases of kcat/Km in dCTP insertion opposite N2-AnthG and N2-BPG (73 and 320-fold) and also kcat/Km in next-base extension from a C paired with each adduct (15 and 51-fold relative to G). Frequencies of dATP misinsertion and extension beyond mispairs were also proportionally increased (70 and 450-fold; 12 and 44-fold) with N2-AnthG and N2-BPG, indicating the effect of adduct bulk on blocking and misincorporation in TLS by pol eta. N2-AnthG and N2-BPG also greatly decreased the pre-steady-state kinetic burst rate (25 and 125-fold) compared to unmodified G. N2-AnthG decreased dCTP binding affinity (2.6-fold) and increased DNA substrate binding affinity. These results and the small kinetic thio effects (S(p)-dCTPalphaS) suggest that the early steps, possibly conformational change, are interfered with by the bulky adducts. In contrast, human pol delta bypassed adducts effectively up to N2-EtG but was strongly blocked by N2-IbG and larger adducts. We conclude that TLS DNA polymerases may be required for the efficient bypass of pol delta-blocking N2-G adducts bulkier than N2-EtG in human cells, and the bulk size can be a major factor for efficient and error-free bypass at these adducts by TLS DNA polymerases.     

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.     

A novel role of DNA polymerase eta in modulating cellular sensitivity to chemotherapeutic agents

Genetic defects in polymerase eta (pol eta; hRad30a gene) result in xeroderma pigmentosum variant syndrome (XP-V), and XP-V patients are sensitive to sunlight and highly prone to cancer development. Here, we show that pol eta plays a significant role in modulating cellular sensitivity to DNA-targeting anticancer agents. When compared with normal human fibroblast cells, pol eta-deficient cells derived from XP-V patients were 3-fold more sensitive to beta-d-arabinofuranosylcytosine, gemcitabine, or cis-diamminedichloroplatinum (cisplatin) single-agent treatments and at least 10-fold more sensitive to the gemcitabine/cisplatin combination treatment, a commonly used clinical regimen for treating a wide spectrum of cancers. Cellular and biochemical analyses strongly suggested that the higher sensitivity of XP-V cells to these agents was due to the inability of pol eta-deficient cells to help resume the DNA replication process paused by the gemcitabine/cisplatin-introduced DNA lesions. These results indicated that pol eta can play an important role in determining the cellular sensitivity to therapeutic agents. The findings not only illuminate pol eta as a potential pharmacologic target for developing new anticancer agents but also provide new directions for improving future chemotherapy regimen design considering the use of nucleoside analogues and cisplatin derivatives.     

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.     

Oxygen free radical damage to DNA. Translesion synthesis by human DNA polymerase eta and resistance to exonuclease action at cyclopurine deoxynucleoside residues.

Cyclopurine deoxynucleosides are common DNA lesions generated by exposure to reactive oxygen species under hypoxic conditions. The S and R diastereoisomers of cyclodeoxyadenosine on DNA were investigated separately for their ability to block 3' to 5' exonucleases. The mammalian DNA-editing enzyme DNase III (TREX1) was blocked by both diastereoisomers, whereas only the S diastereoisomer was highly efficient in preventing digestion by the exonuclease function of T4 DNA polymerase. Digestion in both cases was frequently blocked one residue before the modified base. Oligodeoxyribonucleotides containing a cyclodeoxyadenosine residue were further employed as templates for synthesis by human DNA polymerase eta (pol eta). pol eta could catalyze translesion synthesis on the R diastereoisomer of cyclodeoxyadenosine. On the S diastereoisomer, pol eta could catalyze the incorporation of one nucleotide opposite the lesion but could not continue elongation. Although pol eta preferentially incorporated dAMP opposite the R diastereoisomer, elongation continued only when dTMP was incorporated, suggesting bypass of this lesion by pol eta with reasonable fidelity. With the S diastereoisomer, pol eta mainly incorporated dAMP or dTMP opposite the lesion but could not elongate even after incorporating a correct nucleotide. These data suggest that the S diastereoisomer may be a more cytotoxic DNA lesion than the R diastereoisomer.     

Biochemical basis of genotoxicity of heterocyclic arylamine food mutagens: Human DNA polymerase eta selectively produces a two-base deletion in copying the N2-guanyl adduct of 2-amino-3-methylimidazo[4,5-f]quinoline but not the C8 adduct at the NarI G3 site

Heterocyclic arylamines are highly mutagenic and cause tumors in animal models. The mutagenicity is attributed to the C8- and N2-G adducts, the latter of which accumulates due to slower repair. The C8- and N 2-G adducts derived from 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) were placed at the G1 and G3 sites of the NarI sequence, in which the G3 site is an established hot spot for frameshift mutation with the model arylamine derivative 2-acetylaminofluorene but G1 is not. Human DNA polymerase (pol) eta extended primers beyond template G-IQ adducts better than did pol kappa and much better than pol iota or delta. In 1-base incorporation studies, pol eta inserted C and A, pol iota inserted T, and pol kappa inserted G. Steady-state kinetic parameters were measured for these dNTPs opposite the C8- and N 2-IQ adducts at both sites, being most favorable for pol eta. Mass spectrometry of pol eta extension products revealed a single major product in each of four cases; with the G1 and G3 C8-IQ adducts, incorporation was largely error-free. With the G3 N 2-IQ adduct, a -2 deletion occurred at the site of the adduct. With the G1 N 2-IQ adduct, the product was error-free at the site opposite the base and then stalled. Thus, the pol eta products yielded frame-shifts with the N 2 but not the C8 IQ adducts. We show a role for pol eta and the complexity of different chemical adducts of IQ, DNA position, and DNA polymerases.     

Specificity of DNA lesion bypass by the yeast DNA polymerase eta

DNA polymerase eta (Pol(eta), xeroderma pigmentosum variant, or Rad30) plays an important role in an error-free response to unrepaired UV damage during replication. It faithfully synthesizes DNA opposite a thymine-thymine cis-syn-cyclobutane dimer. We have purified the yeast Pol(eta) and studied its lesion bypass activity in vitro with various types of DNA damage. The yeast Pol(eta) lacked a nuclease or a proofreading activity. It efficiently bypassed 8-oxoguanine, incorporating C, A, and G opposite the lesion with a relative efficiency of approximately 100:56:14, respectively. The yeast Pol(eta) efficiently incorporated a C opposite an acetylaminofluorene-modified G, and efficiently inserted a G or less frequently an A opposite an apurinic/apyrimidinic (AP) site but was unable to extend the DNA synthesis further in both cases. However, some continued DNA synthesis was observed in the presence of the yeast Pol(zeta) following the Pol(eta) action opposite an AP site, achieving true lesion bypass. In contrast, the yeast Pol(alpha) was able to bypass efficiently a template AP site, predominantly incorporating an A residue opposite the lesion. These results suggest that other than UV damage, Pol(eta) may also play a role in bypassing additional DNA lesions, some of which can be error-prone.     

DNA polymerase eta contributes to strand bias of mutations of A versus T in immunoglobulin genes

DNA polymerase (pol) eta participates in hypermutation of A:T bases in Ig genes because humans deficient for the polymerase have fewer substitutions of these bases. To determine whether polymerase eta is also responsible for the well-known preference for mutations of A vs T on the nontranscribed strand, we sequenced variable regions from three patients with xeroderma pigmentosum variant (XP-V) disease, who lack polymerase eta. The frequency of mutations in the intronic region downstream of rearranged J(H)4 gene segments was similar between XP-V and control clones; however, there were fewer mutations of A:T bases and correspondingly more substitutions of C:G bases in the XP-V clones (p < 10(-7)). There was significantly less of a bias for mutations of A compared with T nucleotides in the XP-V clones compared with control clones, whereas the frequencies for mutations of C and G were identical in both groups. An analysis of mutations in the WA sequence motif suggests that polymerase eta generates more mutations of A than T on the nontranscribed strand. This in vivo data from polymerase eta-deficient B cells correlates well with the in vitro specificity of the enzyme. Because polymerase eta inserts more mutations opposite template T than template A, it would generate more substitutions of A on the newly synthesized strand.     

DNA polymerase eta reduces the gamma-H2AX response to psoralen interstrand crosslinks in human cells

DNA interstrand crosslinks are processed by multiple mechanisms whose relationships to each other are unclear. Xeroderma pigmentosum-variant (XP-V) cells lacking DNA polymerase eta are sensitive to psoralen photoadducts created under conditions favoring crosslink formation, suggesting a role for translesion synthesis in crosslink repair. Because crosslinks can lead to double-strand breaks, we monitored phosphorylated H2AX (gamma-H2AX), which is typically generated near double-strand breaks but also in response to single-stranded DNA, following psoralen photoadduct formation in XP-V fibroblasts to assess whether polymerase eta is involved in processing crosslinks. In contrast to conditions favoring monoadducts, conditions favoring psoralen crosslinks induced gamma-H2AX levels in both XP-V and nucleotide excision repair-deficient XP-A cells relative to control repair-proficient cells; ectopic expression of polymerase eta in XP-V cells normalized the gamma-H2AX response. In response to psoralen crosslinking, gamma-H2AX as well as 53BP1 formed coincident foci that were more numerous and intense in XP-V and XP-A cells than in controls. Psoralen photoadducts induced gamma-H2AX throughout the cell cycle in XP-V cells. These results indicate that polymerase eta is important in responding to psoralen crosslinks, and are consistent with a model in which nucleotide excision repair and polymerase eta are involved in processing crosslinks and avoiding gamma-H2AX associated with double-strand breaks and single-stranded DNA in human cells.