Error-prone Translesion Synthesis Research Articles

Near-full-length REV3L appears to be a scarce maternal factor in Xenopus laevis eggs that changes qualitatively in early embryonic development

REV3 is the catalytic subunit of DNA polymerase ζ (pol ζ), which is responsible for the damage-induced mutagenesis that arises during error-prone translesion synthesis in eukaryotes. The related REV3L genes in human and mouse encode proteins of approximately 350 kDa, twice as large as yeast REV3, but full-length REV3L has not been identified in any vertebrate cell. We report that Xenopus laevis REV3L encodes a 352-kDa protein that has high overall amino acid sequence similarity to its mammalian counterparts, and, for the first time in a vertebrate species, we have detected putative REV3L polypeptides of 300 and 340 kDa in X. laevis oocytes. Only the 300-kDa form is stored in eggs, where its concentration of about 65 pM is much lower than those of other replication and repair proteins including the accessory pol ζ subunit REV7. In fertilized eggs, the levels of this polypeptide did not change until neurula; the larger 340-kDa form first appeared at stages after gastrula, suggesting a pattern of regulation during development. These observations indicate the existence of REV3L as a scarce protein, of approximately the full predicted size, whose level may impose severe constraints on the assembly of pol ζ in X. laevis.

Translesion Synthesis of Abasic Sites by Yeast DNA Polymerase ϵ

Studies of replicative DNA polymerases have led to the generalization that abasic sites are strong blocks to DNA replication. Here we show that yeast replicative DNA polymerase epsilon bypasses a model abasic site with comparable efficiency to Pol eta and Dpo4, two translesion polymerases. DNA polymerase epsilon also exhibited high bypass efficiency with a natural abasic site on the template. Translesion synthesis primarily resulted in deletions. In cases where only a single nucleotide was inserted, dATP was the preferred nucleotide opposite the natural abasic site. In contrast to translesion polymerases, DNA polymerase epsilon with 3'-5' proofreading exonuclease activity bypasses only the model abasic site during processive synthesis and cannot reinitiate DNA synthesis. This characteristic may allow other pathways to rescue leading strand synthesis when stalled at an abasic site.

Mechanisms of Dealing with DNA Damage-Induced Replication Problems

During every S phase, cells need to duplicate their genomes so that both daughter cells inherit complete copies of genetic information. Given the large size of mammalian genomes and the required precision of DNA replication, genome duplication requires highly fine-tuned corrective and quality control processes. A major threat to the accuracy and efficiency of DNA synthesis is the presence of DNA lesions, caused by both endogenous and exogenous damaging agents. Replicative DNA polymerases, which carry out the bulk of DNA synthesis, evolved to do their job extremely precisely and efficiently. However, they are unable to use damaged DNA as a template and, consequently, are stopped at most DNA lesions. Failure to restart such stalled replication forks can result in major chromosomal aberrations and lead to cell dysfunction or death. Therefore, a well-coordinated response to replication perturbation is essential for cell survival and fitness. Here we review how this response involves activating checkpoint signaling and the use of specialized pathways promoting replication restart. Checkpoint signaling adjusts cell cycle progression to the emergency situation and thus gives cells more time to deal with the damage. Replication restart is mediated by two pathways. Homologous recombination uses homologous DNA sequence to repair or bypass the lesion and is therefore mainly error free. Error-prone translesion synthesis employs specialized, low fidelity polymerases to bypass the damage.

Modelling the SOS Response by Semi-Stochastic Simulation

The SOS (save our soul) response induced by DNA damage in bacteria E coli has raised a great interests in biophysics and has been extensively studied. Previously we have developed a stochastic simulation model to explain the oscillatory-like modulation of SOS gene expression observed in experiment. Here we present an improved semi-stochastic model which has higher simulation efficiency taking into account the updated knowledge about SOS response. The improved model suggests that frequency of the modulation is controlled by the negative feedback in the system. DNA polymerase V, the key enzyme for error-prone translesion synthesis during SOS response, plays a major role in closing up the negative feedback. It is also indicated that the correlation between the modulation and cellular growth observed in experiment is due to DNA damage induced slowing down of transcription and translation.

DNA Damage-induced Ubiquitylation of RFC2 Subunit of Replication Factor C Complex

Many proteins involved in DNA replication and repair undergo post-translational modifications such as phosphorylation and ubiquitylation. Proliferating cell nuclear antigen (PCNA; a homotrimeric protein that encircles double-stranded DNA to function as a sliding clamp for DNA polymerases) is monoubiquitylated by the RAD6-RAD18 complex and further polyubiquitylated by the RAD5-MMS2-UBC13 complex in response to various DNA-damaging agents. PCNA mono- and polyubiquitylation activate an error-prone translesion synthesis pathway and an error-free pathway of damage avoidance, respectively. Here we show that replication factor C (RFC; a heteropentameric protein complex that loads PCNA onto DNA) was also ubiquitylated in a RAD18-dependent manner in cells treated with alkylating agents or H(2)O(2). A mutant form of RFC2 with a D228A substitution (corresponding to a yeast Rfc4 mutation that reduces an interaction with replication protein A (RPA), a single-stranded DNA-binding protein) was heavily ubiquitylated in cells even in the absence of DNA damage. Furthermore RFC2 was ubiquitylated by the RAD6-RAD18 complex in vitro, and its modification was inhibited in the presence of RPA. The inhibitory effect of RPA on RFC2 ubiquitylation was relatively specific because RAD6-RAD18-mediated ubiquitylation of PCNA was RPA-insensitive. Our findings suggest that RPA plays a regulatory role in DNA damage responses via repression of RFC2 ubiquitylation in human cells.

Formation and genotoxicity of a guanine–cytosine intrastrand cross-link lesion in vivo

Reactive oxygen species (ROS) can be induced by both endogenous and exogenous processes, and they can damage biological molecules including nucleic acids. Exposure of isolated DNA to X/γ-rays and Fenton reagents was shown to lead to the formation of intrastrand cross-link lesions where the neighboring nucleobases in the same DNA strand are covalently bonded. By employing HPLC coupled with tandem mass spectrometry (LC-MS/MS) with the isotope dilution method, we assessed quantitatively the formation of a guanine–cytosine (G[8-5]C) intrastrand cross-link lesion in HeLa-S3 cells upon exposure to γ-rays. The yield of the G[8-5]C cross-link was 0.037 lesions per 109 nucleosides per Gy, which was ∼300 times lower than that of 5-formyl-2′-deoxyuridine (0.011 lesions per 106 nucleosides per Gy) under identical exposure conditions. We further constructed a single-stranded M13 genome harboring a site-specifically incorporated G[8-5]C lesion and developed a novel mass spectrometry-based method for interrogating the products emanating from the replication of the genome in Escherichia coli cells. The results demonstrated that G[8-5]C blocked considerably DNA replication as represented by a 20% bypass efficiency, and the lesion was significantly mutagenic in vivo, which included a 8.7% G→T and a 1.2% G→C transversion mutations. DNA replication in E. coli hosts deficient in SOS-induced polymerases revealed that polymerase V was responsible for the error-prone translesion synthesis in vivo.

Formation of lysine 63-linked poly-ubiquitin chains protects human lung cells against benzo[ a]pyrene-diol-epoxide-induced mutagenicity

Benzo[ a]pyrene exerts its mutagenic effects via induction of benzo[ a]pyrene-diol-epoxide (BPDE)–DNA adducts. Such helix-distorting adducts are not always successfully repaired prior to DNA replication, which may result in a blocked replication fork. To alleviate this stall, cells utilize DNA damage tolerance systems involving either error-free damage avoidance or error-prone translesion synthesis. Studies in yeast suggest the modification of PCNA by lysine 63-linked poly-ubiquitin (K63-polyUb) chains as a key mediator of the error-free damage avoidance pathway. Recently, we extended this observation to human cells, showing the occurrence of poly-ubiquitination of PCNA in UV-irradiated human cells. In the present study, we hypothesized that disrupting the formation of K63-polyUb chains inhibits damage avoidance and favors error-prone repair involving low-fidelity polymerases (e.g. POLη), causing increased BPDE-induced mutagenicity. To test this hypothesis, we generated A549 cells expressing either a mutant ubiquitin ( K63R- Ub) which blocks further ubiquitination through K63, or the wild type ubiquitin ( WT- Ub). We show that PCNA is poly-ubiquitinated in these cells upon BPDE-exposure and that disruption of K63-polyUb chain formation has no effect on BPDE-induced toxicity. In contrast, significantly higher frequencies of BPDE-induced HPRT mutations were observed in K63R- Ub expressing cells, of which the majority (74%) was G → T transversion. BPDE treatment caused an enhanced recruitment of POLη to the replication machinery of the K63R- Ub expressing cells, where it co-localized with PCNA. Suppression of POLη expression by using siRNA resulted in a 50% reduction of BPDE-induced mutations in the K63R cells. In conclusion, we demonstrated that formation of K63-polyUb chains protects BPDE-exposed human cells against translesion synthesis-mediated mutagenesis. These findings indicate that K63-polyubiquitination guards against chemical carcinogenesis by preventing mutagenesis and thus contributing to genomic stability.

Regulation of monoubiquitinated PCNA by DUB autocleavage

Monoubiquitination is a reversible post-translational protein modification that has an important regulatory function in many biological processes, including DNA repair. Deubiquitinating enzymes (DUBs) are proteases that are negative regulators of monoubiquitination, but little is known about their regulation and contribution to the control of conjugated-substrate levels. Here, we show that the DUB ubiquitin specific protease 1 (USP1) deubiquitinates the DNA replication processivity factor, PCNA, as a safeguard against error-prone translesion synthesis (TLS) of DNA. Ultraviolet (UV) irradiation inactivates USP1 through an autocleavage event, thus enabling monoubiquitinated PCNA to accumulate and to activate TLS. Significantly, the site of USP1 cleavage is immediately after a conserved internal ubiquitin-like diglycine (Gly-Gly) motif. This mechanism is reminiscent of the processing of precursors of ubiquitin and ubiquitin-like modifiers by DUBs. Our results define a regulatory mechanism for protein ubiquitination that involves the signal-induced degradation of an inhibitory DUB.

Translesion synthesis DNA polymerases and control of genome stability

Eukaryotic and prokaryotic genomes are replicated with amazingly high fidelity to assure faithful transmission of genetic information from one generation to the next. The accuracy of replication relies heavily on the ability of replicative DNA polymerases to efficiently select correct nucleotides for the polymerization reaction and excise mistakenly incorporated nucleotides using their intrinsic exonucleases. Cell also possess a variety of specialized DNA polymerases that help to overcome replication blocks when occasional unrepaired DNA lesions stall the replication machinery. The translesion synthesis (TLS) polymerases have an extremely low fidelity during copying undamaged DNA substrates, such that uncontrolled participation of these polymerases in DNA replication could present a threat to the genome stability. In this article, we discuss the properties of prokaryotic and eukaryotic TLS polymerases and their roles in modulating the rate of spontaneous and genotoxicant-induced mutations. We also review recent insights into the molecular mechanisms regulating the participation of error-prone TLS polymerases in the genome replication. Finally, we discuss the relationship between the functions of TLS polymerases and human disease.

Error-prone Translesion Synthesis by Human DNA Polymerase η on DNA-containing Deoxyadenosine Adducts of 7,8-Dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene

When human DNA polymerase eta (pol eta) encounters N6-deoxyadenosine adducts formed by trans epoxide ring opening of the 7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BaP DE) isomer with (+)-7R,8S,9S,10R configuration ((+)-BaP DE-2), misincorporation of A or G and incorporation of the correct T are equally likely to occur. On the other hand, the enzyme exhibits a 3-fold preference for correct T incorporation opposite adducts formed by trans ring opening of the (-)-(7S,8R,9R,10S)-DE-2 enantiomer. Adducts at dA formed by cis ring opening of these two BaP DE-2 isomers exhibit a 2-3-fold preference for A over T incorporation, with G intermediate between the two. Extension one nucleotide beyond these adducts is generally weaker than incorporation across from them, but among mismatches the (adducted A*) x A mispair is the most favored for extension. Because mutations can only occur if mispairs are extended, this observation is consistent with the occurrence of A x T to T x A transversions as common mutations in animal cells treated with BaP DE-2 isomers. Adducts with S absolute configuration at the point of attachment of the hydrocarbon to the base inhibit incorporation and extension by pol eta to a lesser extent than their R counterparts. Template-primers containing each of the four isomeric dA adducts derived from BaP DE-2 and two adducts derived from 9,10-epoxy-7,8,9,10-tetrahydrobenzo-[a]pyrene in which the 7- and 8-hydroxyl groups of the DEs are replaced with hydrogens exhibit reduced electrophoretic mobilities relative to the unadducted oligonucleotides. This effect is largely independent of DNA sequence. Decreased mobility correlates with an increased rate of incorporation by pol eta, suggesting a systematic relationship between the overall DNA structure and efficiency of the enzyme.

The p-benzoquinone DNA adducts derived from benzene are highly mutagenic

Benzene is a human leukemia carcinogen, resulting from its cellular metabolism. A major benzene metabolite is p-benzoquinone (pBQ), which can damage DNA by forming the exocyclic base adducts pBQ-dC, pBQ-dA, and pBQ-dG in vitro. To gain insights into the role of pBQ in benzene genotoxicity, we examined in vitro translesion synthesis and in vivo mutagenesis of these pBQ adducts. Purified REV1 and Polκ were essentially incapable of translesion synthesis in response to the pBQ adducts. Opposite pBQ-dA and pBQ-dC, purified human Polι was capable of error-prone nucleotide insertion, but was unable to perform extension synthesis. Error-prone translesion synthesis was observed with Polη. However, DNA synthesis largely stopped opposite the lesion. Consistent with in vitro results, replication of site-specifically damaged plasmids was strongly inhibited by pBQ adducts in yeast cells, which depended on both Polζ and Polη. In wild-type cells, the majority of translesion products were deletions at the site of damage, accounting for 91%, 90%, and 76% for pBQ-dA, pBQ-dG, and pBQ-dC, respectively. These results show that the pBQ-dC, pBQ-dA, and pBQ-dG adducts are strong blocking lesions, and are highly mutagenic by predominantly inducing deletion mutations. These results are consistent with the lesion structures predicted by molecular dynamics simulation. Our results led to the following model. Translesion synthesis normally occurs by directly copying the lesion site through base insertion and extension synthesis. When the lesion becomes incompatible in accommodating a base opposite the lesion in DNA, translesion synthesis occurs by a less efficient lesion loop-out mechanism, resulting in avoiding copying the damaged base and leading to deletion.

Epistatic participation of REV1 and REV3 in the formation of UV-induced frameshift mutations in cell cycle-arrested yeast cells

Mutations arising in times of cell cycle arrest may provide a selective advantage for unicellular organisms adapting to environmental changes. For multicellular organisms, however, they may pose a serious threat, in that such mutations in somatic cells contribute to carcinogenesis and ageing. The budding yeast Saccharomyces cerevisiae presents a convenient model system for studying the incidence and the mechanisms of stationary-phase mutation in a eukaryotic organism. Having studied the emergence of frameshift mutants after several days of starvation-induced cell cycle arrest, we previously reported that all (potentially error-prone) translesion synthesis (TLS) enzymes identified in S. cerevisiae did not contribute to the basal level of spontaneous stationary-phase mutations. However, we observed that an increased frequency of stationary-phase frameshift mutations, brought about by a defective nucleotide excision repair (NER) pathway or by UV irradiation, was dependent on Rev3p, the catalytic subunit of the TLS polymerase zeta (Pol ζ). Employing the same two conditions, we now examined the effect of deletions of the genes coding for polymerase eta (Pol η) ( RAD30) and Rev1p ( REV1). In a NER-deficient strain background, the increased incidence of stationary-phase mutations was only moderately influenced by a lack of Pol η but completely reduced to wild type level by a knockout of the REV1 gene. UV-induced stationary-phase mutations were abundant in wild type and rad30Δ strains, but substantially reduced in a rev1Δ as well as a rev3Δ strain. The similarity of the rev1Δ and the rev3Δ phenotype and an epistatic relationship evident from experiments with a double-deficient strain suggests a participation of Rev1p and Rev3p in the same mutagenic pathway. Based on these results, we propose that the response of cell cycle-arrested cells to an excess of exo- or endogenously induced DNA damage includes a novel replication-independent cooperative function of Rev1p and Pol ζ, which has the potential to generate mutations.

Error-prone repair and translesion synthesis III: The activation of UmuD (or less is more)

Following DNA damage to Escherichia coli bacteria, RecA protein is activated by binding to single stranded DNA and cleaves its own gene repressor (LexA protein). Two papers from Graham Walker's laboratory showed that several bacterial genes in addition to RecA are repressed by the LexA repressor and are inducible following DNA damage [C.J. Keyon, G.C. Walker, DNA-damaging agents stimulate gene expression at specific loci in Escherichia coli, in: Proceedings of the National Academy of Sciences of the United States of America 77, 1980, pp. 2819–2823] and predicted that one of them (UmuD) might itself be subject to activation by a further cleavage reaction involving activated RecA protein [K.L. Perry, S.J. Elledge, B.B. Mitchell, L. Marsh, G.C. Walker, umuD,C and mucA,B operans whose products are required for UV light- and chemical-induced mutagenesis: UmuD, MucA, and LexA proteins share homology, in: Proceedings of the National Academy of Sciences of the United States of America 82, 1985, pp. 4331–4335]. The processed form of UmuD, termed UmuD’, later proved to be a subunit of DNA polymerase V, a key enzyme involved in translesion synthesis.

Error-prone DNA repair and translesion synthesis: Focus on the replication fork

Dean Rupp and Paul Howard-Flanders showed that, following exposure to ultraviolet light, bacteria deficient in nucleotide excision repair synthesised DNA with minimal delay and in pieces roughly the size of the distances between pyrimidine dimmers. The discontinuities or gaps between these pieces were subsequently sealed. This led directly to the hypothesis of translesion synthesis.

Accommodation of a 1S-(−)-Benzo[c]phenanthrenyl-N6-dA Adduct in the Y-Family Dpo4 DNA Polymerase Active Site: Structural Insights through Molecular Dynamics Simulations

Molecular modeling and molecular dynamics simulations have been performed to elucidate feasible structures in the Y-family Dpo4 DNA polymerase for the 1S-(-)-trans-anti-B[c]Ph-N6-dA adduct, derived from the fjord region polycyclic aromatic hydrocarbon (PAH) benzo[c]phenanthrene. Three types of models were delineated as follows: an intercalation model, a model with the aromatic ring system in the polymerase major groove open pocket, and a -1 deletion major groove model. All four 2'-deoxyribonucleoside 5'-triphosphates (dNTPs) were considered in the first two cases, and a normal Watson-Crick partner positioned to have skipped the modified template was employed as the incoming dNTP in the -1 deletion case. The trajectories derived from the dynamics simulations were analyzed in detail to evaluate the extents of distortion for each system. Overall, our results suggest that the major groove model is the least distorted, followed by the -1 deletion model, while the intercalation model is perturbed the most. The syn-dGTP and syn-dATP mismatches opposite the lesion are well-accommodated in the major groove model, as is the normal Watson-Crick partner dTTP. The intercalation model appears most likely to impede the polymerase. More broadly, these models look reasonable for other PAH metabolite-derived adducts to adenine with similar 1S stereochemistry. Furthermore, these models suggest how error-prone translesion synthesis by Y-family polymerases might produce mutations that may play a role in the initiation of cancer.

The BRCT domain of mammalian Rev1 is involved in regulating DNA translesion synthesis

Rev1 is a deoxycytidyl transferase associated with DNA translesion synthesis (TLS). In addition to its catalytic domain, Rev1 possesses a so-called BRCA1 C-terminal (BRCT) domain. Here, we describe cells and mice containing a targeted deletion of this domain. Rev1B/B mice are healthy, fertile and display normal somatic hypermutation. Rev1B/B cells display an elevated spontaneous frequency of intragenic deletions at Hprt. In addition, these cells were sensitized to exogenous DNA damages. Ultraviolet-C (UV-C) light induced a delayed progression through late S and G2 phases of the cell cycle and many chromatid aberrations, specifically in a subset of mutant cells, but not enhanced sister chromatid exchanges (SCE). UV-C-induced mutagenesis was reduced and mutations at thymidine–thymidine dimers were absent in Rev1B/B cells, the opposite phenotype of UV-C-exposed cells from XP-V patients, lacking TLS polymerase η. This suggests that the enhanced UV-induced mutagenesis in XP-V patients may depend on error-prone Rev1-dependent TLS. Together, these data indicate a regulatory role of the Rev1 BRCT domain in TLS of a limited spectrum of endogenous and exogenous nucleotide damages during a defined phase of the cell cycle.

The role of DNA polymerase eta in translesion synthesis past platinum-DNA adducts in human fibroblasts.

Cisplatin, a widely used chemotherapeutic agent, has been implicated in the induction of secondary tumors in cancer patients. This drug is presumed to be mutagenic because of error-prone translesion synthesis of cisplatin adducts in DNA. Oxaliplatin is effective in cisplatin-resistant tumors, but its mutagenicity in humans has not been reported. The polymerases involved in bypass of cisplatin and oxaliplatin adducts in vivo are not known. DNA polymerase eta is the most efficient polymerase for bypassing platinum adducts in vitro. We evaluated the role of polymerase eta in translesion synthesis past platinum adducts by determining cytotoxicity and induced mutation frequencies at the hypoxanthine guanine phosphoribosyltransferase (HPRT) locus in diploid human fibroblasts. Normal human fibroblasts (NHF1) were compared with xeroderma pigmentosum variant (XPV) cells (polymerase eta-null) after treatment with cisplatin. In addition, XPV cells complemented for polymerase eta expression were compared with the isogenic cells carrying the empty expression vector. Cytotoxicity and induced mutagenicity experiments were measured in parallel in UVC-irradiated fibroblasts. We found that equitoxic doses of cisplatin induced mutations in fibroblasts lacking polymerase eta at frequencies 2- to 2.5-fold higher than in fibroblasts with either normal or high levels of polymerase eta. These results indicate that polymerase eta is involved in error-free translesion synthesis past some cisplatin adducts. We also found that per lethal event, cisplatin was less mutagenic than UVC. Treatment with a wide range of cytotoxic doses of oxaliplatin did not induce mutations above background levels in cells either expressing or lacking polymerase eta, suggesting that oxaliplatin is nonmutagenic in human fibroblasts.

In vitro mutation artifacts after formalin fixation and error prone translesion synthesis during PCR.

BackgroundClinical specimens are routinely fixed in 10% buffered formalin and paraffin embedded. Although DNA is commonly extracted from fixed tissues and amplified by PCR, the effects of formalin fixation are relatively unknown. Formalin fixation is known to impair PCR, presumably through damage that blocks polymerase elongation, but an insidious possibility is error prone translesion synthesis across sites of damage, producing in vitro artifactual mutations during PCR.MethodsTo better understand the consequences of fixation, DNA specimens extracted from fresh or fixed tissues were amplified with Taq DNA polymerase, and their PCR products were cloned and sequenced.ResultsSignificantly more (3- to 4-fold) mutations were observed with fixed DNA specimens. The majority of mutations were transitions, predominantly at A:T base pairs, randomly distributed along the template.ConclusionsFormalin fixation appears to cause random base damage, which can be bridged during PCR by Taq DNA polymerase through error prone translesion synthesis. Fixed DNA is a damaged but "readable" template.

Translesion synthesis of acetylaminofluorene-dG adducts by DNA polymerase zeta is stimulated by yeast Rev1 protein.

Translesion synthesis is an important mechanism in response to unrepaired DNA lesions during replication. The DNA polymerase zeta (Polzeta) mutagenesis pathway is a major error-prone translesion synthesis mechanism requiring Polzeta and Rev1. In addition to its dCMP transferase, a non-catalytic function of Rev1 is suspected in cellular response to certain types of DNA lesions. However, it is not well understood about the non-catalytic function of Rev1 in translesion synthesis. We have analyzed the role of Rev1 in translesion synthesis of an acetylaminofluorene (AAF)-dG DNA adduct. Purified yeast Rev1 was essentially unresponsive to a template AAF-dG DNA adduct, in contrast to its efficient C insertion opposite a template 1,N6-ethenoadenine adduct. Purified yeast Polzeta was very inefficient in the bypass of the AAF-dG adduct. Combining Rev1 and Polzeta, however, led to a synergistic effect on translesion synthesis. Rev1 protein enhanced Polzeta-catalyzed nucleotide insertion opposite the AAF-dG adduct and strongly stimulated Polzeta-catalyzed extension from opposite the lesion. Rev1 also stimulated the deficient synthesis by Polzeta at the very end of undamaged DNA templates. Deleting the C-terminal 205 aa of Rev1 did not affect its dCMP transferase activity, but abolished its stimulatory activity on Polzeta-catalyzed extension from opposite the AAF-dG adduct. These results suggest that translesion synthesis of AAF-dG adducts by Polzeta is stimulated by Rev1 protein in yeast. Consistent with the in vitro results, both Polzeta and Rev1 were found to be equally important for error-prone translesion synthesis across from AAF-dG DNA adducts in yeast cells.

Mutagenesis of benzo[a]pyrene diol epoxide in yeast: requirement for DNA polymerase zeta and involvement of DNA polymerase eta.

Benzo[a]pyrene is a potent environmental carcinogen, which can be metabolized in cells to the DNA damaging agent anti-benzo[a]pyrene-7,8-dihydrodiol-9,10-epoxide (anti-BPDE). We hypothesize that mutations induced by BPDE DNA adducts are mainly generated through an error-prone translesion synthesis that requires a specialized DNA polymerase (Pol). Using an in vivo mutagenesis assay in the yeast model system, we have examined the potential roles of Pol(zeta) and Pol(eta) in (+/-)-anti-BPDE-induced mutagenesis. In cells proficient in mutagenesis, (+/-)-anti-BPDE induced 85% base substitutions with predominant G --> C followed by G --> T transversions, 9% deletions of 1-3 nucleotides, and 6% insertions of 1-3 nucleotides. In rad30 mutant cells lacking Pol(eta), (+/-)-anti-BPDE-induced mutagenesis was reduced and accompanied by a moderate decrease in base substitutions and more significant decrease in deletions and insertions of 1-3 nucleotides. In rev3 mutant cells lacking Pol(zeta), (+/-)-anti-BPDE-induced mutagenesis was mostly abolished, leading to a great decrease in both base substitutions and deletions/insertions of 1-3 nucleotides. In contrast, large deletions/insertions were significantly increased in cells lacking Pol(zeta). Consistent with the in vivo results, purified yeast Pol(zeta) performed limited translesion synthesis opposite (+)- and (-)-trans-anti-BPDE-N(2)-dG DNA adducts with predominant G incorporation opposite the lesion. These results show that (+/-)-anti-BPDE-induced mutagenesis in yeast requires Pol(zeta) and partially involves Pol(eta) and suggest that Pol(zeta) directly participates in nucleotide insertions opposite the lesion, while Pol(eta) significantly contributes to deletions and insertions of 1-3 nucleotides.