Translesion Synthesis DNA Polymerases Research Articles

Rad5 Recruits Error-Prone DNA Polymerases for Mutagenic Repair of ssDNA Gaps on Undamaged Templates

Post-replication repair (PRR) allows tolerance of chemical- and UV-induced DNA base lesions in both an error-free and an error-prone manner. In classical PRR, PCNA monoubiquitination recruits translesion synthesis (TLS) DNA polymerases that can replicate through lesions. We find that PRR responds to DNA replication stress that does not cause base lesions. Rad5 forms nuclear foci during normal S phase and after exposure to types of replication stress where DNA base lesions are likely absent. Rad5 binds to the sites of stressed DNA replication forks, where it recruits TLS polymerases to repair single-stranded DNA (ssDNA) gaps, preventing mitotic defects and chromosome breaks. In contrast to the prevailing view of PRR, our data indicate that Rad5 promotes both mutagenic and error-free repair of undamaged ssDNA that arises during physiological and exogenous replication stress.

AKT inhibition impairs PCNA ubiquitylation and triggers synthetic lethality in homologous recombination-deficient cells submitted to replication stress

Translesion DNA synthesis (TLS) and homologous recombination (HR) cooperate during S-phase to safeguard replication forks integrity. Thus, the inhibition of TLS becomes a promising point of therapeutic intervention in HR-deficient cancers, where TLS impairment might trigger synthetic lethality (SL). The main limitation to test this hypothesis is the current lack of selective pharmacological inhibitors of TLS. Herein, we developed a miniaturized screening assay to identify inhibitors of PCNA ubiquitylation, a key post-translational modification required for efficient TLS activation. After screening a library of 627 kinase inhibitors, we found that targeting the pro-survival kinase AKT leads to strong impairment of PCNA ubiquitylation. Mechanistically, we found that AKT-mediated modulation of Proliferating Cell Nuclear Antigen (PCNA) ubiquitylation after UV requires the upstream activity of DNA PKcs, without affecting PCNA ubiquitylation levels in unperturbed cells. Moreover, we confirmed that persistent AKT inhibition blocks the recruitment of TLS polymerases to sites of DNA damage and impairs DNA replication forks processivity after UV irradiation, leading to increased DNA replication stress and cell death. Remarkably, when we compared the differential survival of HR-proficient vs HR-deficient cells, we found that the combination of UV irradiation and AKT inhibition leads to robust SL induction in HR-deficient cells. We link this phenotype to AKT ability to inhibit PCNA ubiquitylation, since the targeted knockdown of PCNA E3-ligase (RAD18) and a non-ubiquitylable (PCNA K164R) knock-in model recapitulate the observed SL induction. Collectively, this work identifies AKT as a novel regulator of PCNA ubiquitylation and provides the proof-of-concept of inhibiting TLS as a therapeutic approach to selectively kill HR-deficient cells submitted to replication stress.

Structural Approach To Identify a Lead Scaffold That Targets the Translesion Synthesis Polymerase Rev1.

Translesion synthesis (TLS) is a mechanism of replication past damaged DNA through which multiple forms of human cancer survive and acquire resistance to first-line genotoxic chemotherapies. As such, TLS is emerging as a promising target for the development of a new class of anticancer agents. The C-terminal domain of the DNA polymerase Rev1 (Rev1-CT) mediates assembly of the functional TLS complex through protein-protein interactions (PPIs) with Rev1 interacting regions (RIRs) of several other TLS DNA polymerases. Utilizing structural knowledge of the Rev1-CT/RIR interface, we have identified the phenazopyridine scaffold as an inhibitor of this essential TLS PPI. We demonstrate direct binding of this scaffold to Rev1-CT, and the synthesis and evaluation of a small series of analogues have provided important structure-activity relationships for further development of this scaffold. Furthermore, we utilized the umbrella sampling method to predict the free energy of binding to Rev1-CT for each of our analogues. Binding energies calculated through umbrella sampling correlated well with experimentally determined IC50 values, validating this computational tool as a viable approach to predict the biological activity for inhibitors of the Rev1-CT/RIR PPI.

Cytotoxic and mutagenic properties of O6-alkyl-2′-deoxyguanosine lesions in Escherichia coli cells

Environmental exposure and cellular metabolism can give rise to DNA alkylation, which can occur on the nitrogen and oxygen atoms of nucleobases, as well as on the phosphate backbone. Although O6-alkyl-2'-deoxyguanosine (O6-alkyl-dG) lesions are known to be associated with cancer, not much is known about how the alkyl group structures in these lesions affect their repair and replicative bypass in vivo or how translesion synthesis DNA polymerases influence the latter process. To answer these questions, here we synthesized oligodeoxyribonucleotides harboring seven O6-alkyl-dG lesions, with the alkyl group being Me, Et, nPr, iPr, nBu, iBu, or sBu, and examined the impact of these lesions on DNA replication in Escherichia coli cells. We found that replication past all the O6-alkyl-dG lesions was highly efficient and that SOS-induced DNA polymerases play redundant roles in bypassing these lesions. Moreover, these lesions directed exclusively the G → A mutation, the frequency of which increased with the size of the alkyl group on the DNA. This could be attributed to the varied repair efficiencies of these lesions by O6-alkylguanine DNA alkyltransferase (MGMT) in cells, which involve the MGMT Ogt and, to a lesser extent, Ada. In conclusion, our study provides important new knowledge about the repair of the O6-alkyl-dG lesions and their recognition by the E. coli DNA replication machinery. Our results suggest that the lesions' carcinogenic potentials may be attributed, at least in part, to their strong mutagenic potential and their efficient bypass by the DNA replication machinery.

Structural basis for polymerase \u03b7\u2013promoted resistance to the anticancer nucleoside analog cytarabine

Cytarabine (AraC) is an essential chemotherapeutic for acute myeloid leukemia (AML) and resistance to this drug is a major cause of treatment failure. AraC is a nucleoside analog that differs from 2′-deoxycytidine only by the presence of an additional hydroxyl group at the C2′ position of the 2′-deoxyribose. The active form of the drug AraC 5′-triphosphate (AraCTP) is utilized by human replicative DNA polymerases to insert AraC at the 3′ terminus of a growing DNA chain. This impedes further primer extension and is a primary basis for the drug action. The Y-family translesion synthesis (TLS) DNA polymerase η (Polη) counteracts this barrier to DNA replication by efficient extension from AraC-terminated primers. Here, we provide high-resolution structures of human Polη with AraC incorporated at the 3′-primer terminus. We show that Polη can accommodate AraC at different stages of the catalytic cycle, and that it can manipulate the conformation of the AraC sugar via specific hydrogen bonding and stacking interactions. Taken together, the structures provide a basis for the ability of Polη to extend DNA synthesis from AraC terminated primers.

Impact of tobacco-specific nitrosamine–derived DNA adducts on the efficiency and fidelity of DNA replication in human cells

The tobacco-derived nitrosamines 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and N'-nitrosonornicotine (NNN) are known human carcinogens. Following metabolic activation, NNK and NNN can induce a number of DNA lesions, including several 4-(3-pyridyl)-4-oxobut-1-yl (POB) adducts. However, it remains unclear to what extent these lesions affect the efficiency and accuracy of DNA replication and how their replicative bypass is influenced by translesion synthesis (TLS) DNA polymerases. In this study, we investigated the effects of three stable POB DNA adducts (O2-POB-dT, O4-POB-dT, and O6-POB-dG) on the efficiency and fidelity of DNA replication in HEK293T human cells. We found that, when situated in a double-stranded plasmid, O2-POB-dT and O4-POB-dT moderately blocked DNA replication and induced exclusively T→A (∼14.9%) and T→C (∼35.2%) mutations, respectively. On the other hand, O6-POB-dG slightly impeded DNA replication, and this lesion elicited primarily the G→A transition (∼75%) together with a low frequency of the G→T transversion (∼3%). By conducting replication studies in isogenic cells in which specific TLS DNA polymerases (Pols) were deleted by CRISPR-Cas9 genome editing, we observed that multiple TLS Pols, especially Pol η and Pol ζ, are involved in bypassing these lesions. Our findings reveal the cytotoxic and mutagenic properties of specific POB DNA adducts and unravel the roles of several TLS polymerases in the replicative bypass of these adducts in human cells. Together, these results provide important new knowledge about the biological consequences of POB adducts.

5-Formylcytosine mediated DNA–protein cross-links block DNA replication and induce mutations in human cells

5-Formylcytosine (5fC) is an epigenetic DNA modification introduced via TET protein-mediated oxidation of 5-methyl-dC. We recently reported that 5fC form reversible DNA–protein conjugates (DPCs) with histone proteins in living cells (Ji et al. (2017) Angew. Chem. Int. Ed., 56:14130–14134). We now examined the effects of 5fC mediated DPCs on DNA replication. Synthetic DNA duplexes containing site-specific DPCs between 5fC and lysine-containing proteins and peptides were subjected to primer extension experiments in the presence of human translesion synthesis DNA polymerases η and κ. We found that DPCs containing histones H2A or H4 completely inhibited DNA replication, but the replication block was removed when the proteins were subjected to proteolytic digestion. Cross-links to 11-mer or 31-mer peptides were bypassed by both polymerases in an error-prone manner, inducing targeted C→T transitions and –1 deletions. Similar types of mutations were observed when plasmids containing 5fC-peptide cross-links were replicated in human embryonic kidney (HEK) 293T cells. Molecular simulations of the 11-mer peptide-dC cross-links bound to human polymerases η and κ revealed that the peptide fits well on the DNA major groove side, and the modified dC forms a stable mismatch with incoming dATP via wobble base pairing in the polymerase active site.

Polymerase Delta in Eukaryotes: How is It Transiently Exchanged with Specialized DNA Polymerases During Translesion DNA Synthesis?

Precise duplication of the human genome is constantly threatened by a variety of genotoxic insults. During S-phase, those damaged template bases could be overcome by DNA damage tolerance (DDT) pathways that bypass such obstacles instead of repairing them, allowing replicative machinery to resume beyond the offending lesions. Two distinct strategies of DDT, template switching and translesion DNA synthesis (TLS), are employed in eukaryotes. In the former process, the newly synthesized sister chromatid is utilized as an undamaged template to restart recombination-dependent DNA synthesis in an error-free manner. While TLS process involves a reversible polymerase switching between replicative and specialized TLS polymerases for the rescue of stalled replication forks, but this process is intrinsically error-prone and thereby increases mutation rates that potentially drive cancer and aging. It still remains controversial on what exact molecular mechanism orchestrates the polymerase switching at blocked primer-template (P/T) junction. In this review, we summarize and discuss the details of multiple types of mechanisms on how DNA polymerase switching is coordinated during TLS in eukaryotic systems. We also propose a hypothesis regarding high-fidelity human DNA polymerase delta (pol δ) and its involvement in polymerase switching based on recent progress in its functional and structural characterization, especially post-translational modification of its subunits, to gain further insights into the intriguing mechanisms of its regulation during TLS.

Cytotoxic and mutagenic properties of minor-groove O2-alkylthymidine lesions in human cells

Endogenous metabolism, environmental exposure, and cancer chemotherapy can lead to alkylation of DNA. It has been well documented that, among the different DNA alkylation products, minor-groove O2-alkylthymidine (O2-alkyldT) lesions are inefficiently repaired. In the present study, we examined how seven O2-alkyldT lesions, with the alkyl group being a Me, Et, nPr, iPr, nBu, iBu, or sBu, are recognized by the DNA replication machinery in human cells. We found that the replication bypass efficiencies of these lesions decrease with increasing length of the alkyl chain, and that these lesions induce substantial frequencies of T→A and T→G mutations. Replication experiments using isogenic cells deficient in specific translesion synthesis (TLS) DNA polymerases revealed that the absence of polymerase η or polymerase ζ, but not polymerase κ or polymerase ι, significantly decreased both the bypass efficiencies and the mutation frequencies for those O2-alkyldT lesions carrying a straight-chain alkyl group. Moreover, the mutagenic properties of the O2-alkyldT lesions were influenced by the length and topology of the alkyl chain and by TLS polymerases. Together, our results provide important new knowledge about the cytotoxic and mutagenic properties of O2-alkyldT lesions, and illustrate the roles of TLS polymerases in replicative bypass of these lesions in human cells.

Structural Basis for the Interaction of Mutasome Assembly Factor REV1 with Ubiquitin

REV1 is an evolutionarily conserved translesion synthesis (TLS) DNA polymerase and an assembly factor key for the recruitment of other TLS polymerases to DNA damage sites. REV1-mediated recognition of ubiquitin in the proliferative cell nuclear antigen is thought to be the trigger for TLS activation. Here we report the solution NMR structure of a 108-residue fragment of human REV1 encompassing the two putative ubiquitin-binding motifs UBM1 and UBM2 in complex with ubiquitin. While in mammals UBM1 and UBM2 are both required for optimal association of REV1 with replication factories after DNA damage, we show that only REV1 UBM2 binds ubiquitin. Structure-guided mutagenesis in Saccharomyces cerevisiae further highlights the importance of UBM2 for REV1-mediated mutagenesis and DNA damage tolerance.

Escherichia coli RarA commits cells to post‐replication repair pathways by facilitating replisome skipping

Members of the highly conserved RarA/Mgs1/WRNIP1 protein family have been implicated in the maintenance of genomic stability, but their exact functions remain unknown. Loss of one of these proteins rarely results in an altered phenotype, but a knockout mutation in genes encoding proteins of this family in tandem with the loss of other DNA metabolism functions can adversely affect cell survival. We hypothesize that the RarA protein of E. coli commits the cell to post‐replication repair pathways, namely translesion DNA synthesis (TLS) and daughter strand gap synthesis. RarA overexpression is toxic to the cell and knocking out DNA Polymerase IV and DNA Polymerase V, TLS‐competent polymerases, alleviates this toxicity. Furthermore, the presence of RarA induces sensitivity in cells lacking TLS polymerases to the DNA‐damaging agents nitrofurazone (NFZ) and 4‐nitroquinoline‐1‐oxide (4‐NQO). Deletion of RarA suppresses DNA Polymerase IV mediated sensitivity to NFZ and 4‐NQO. These data suggest that RarA can promote rapid replisome progress by committing cells to using post‐replication repair pathways and blocking access to other repair pathways when the replisome encounters certain types of lesions.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Cytotoxic and mutagenic properties of alkyl phosphotriester lesions in Escherichia coli cells.

Exposure to many endogenous and exogenous agents can give rise to DNA alkylation, which constitutes a major type of DNA damage. Among the DNA alkylation products, alkyl phosphotriesters have relatively high frequencies of occurrence and are resistant to repair in mammalian tissues. However, little is known about how these lesions affect the efficiency and fidelity of DNA replication in cells or how the replicative bypass of these lesions is modulated by translesion synthesis DNA polymerases. In this study, we synthesized oligodeoxyribonucleotides containing four pairs (Sp and Rp) of alkyl phosphotriester lesions at a defined site, and examined how these lesions are recognized by DNA replication machinery in Escherichia coli cells. We found that the Sp diastereomer of the alkyl phosphotriester lesions could be efficiently bypassed, whereas the Rp counterparts moderately blocked DNA replication. Moreover, the Sp-methyl phosphotriester induced TT→GT and TT→GC mutations at the flanking TT dinucleotide site, and the induction of these mutations required Ada protein, which is known to remove efficiently the methyl group from the Sp-methyl phosphotriester. Together, our study provided a comprehensive understanding about the recognition of alkyl phosphotriester lesions by DNA replication machinery in cells, and revealed for the first time the Ada-dependent induction of mutations at the Sp-methyl phosphotriester site.

Genetic control of predominantly error-free replication through an acrolein-derived minor-groove DNA adduct

Acrolein, an α,β-unsaturated aldehyde, is generated in vivo as the end product of lipid peroxidation and from metabolic oxidation of polyamines, and it is a ubiquitous environmental pollutant. The reaction of acrolein with the N2 of guanine in DNA leads to the formation of γ-hydroxy-1-N2-propano-2' deoxyguanosine (γ-HOPdG), which can exist in DNA in a ring-closed or a ring-opened form. Here, we identified the translesion synthesis (TLS) DNA polymerases (Pols) that conduct replication through the permanently ring-opened reduced form of γ-HOPdG ((r) γ-HOPdG) and show that replication through this adduct is mediated via Rev1/Polη-, Polι/Polκ-, and Polθ-dependent pathways, respectively. Based on biochemical and structural studies, we propose a role for Rev1 and Polι in inserting a nucleotide (nt) opposite the adduct and for Pols η and κ in extending synthesis from the inserted nt in the respective TLS pathway. Based on genetic analyses and biochemical studies with Polθ, we infer a role for Polθ at both the nt insertion and extension steps of TLS. Whereas purified Rev1 and Polθ primarily incorporate a C opposite (r) γ-HOPdG, Polι incorporates a C or a T opposite the adduct; nevertheless, TLS mediated by the Polι-dependent pathway as well as by other pathways occurs in a predominantly error-free manner in human cells. We discuss the implications of these observations for the mechanisms that could affect the efficiency and fidelity of TLS Pols.

Role of Translesion Synthesis DNA Polymerases in DNA Replication in the Presence of a Weak DNA Polymerase δ in Saccharomyces cerevisiae

Role of Translesion Synthesis DNA Polymerases in DNA Replication in the Presence of a Weak DNA Polymerase δ in Saccharomyces cerevisiae

The C-terminal region of translesion synthesis DNA polymerase η is partially unstructured and has high conformational flexibility.

Eukaryotic DNA polymerase η catalyzes translesion synthesis of thymine dimers and 8-oxoguanines. It is comprised of a polymerase domain and a C-terminal region, both of which are required for its biological function. The C-terminal region mediates interactions with proliferating cell nuclear antigen (PCNA) and other translesion synthesis proteins such as Rev1. This region contains a ubiquitin-binding/zinc-binding (UBZ) motif and a PCNA-interacting protein (PIP) motif. Currently little structural information is available for this region of polymerase η. Using a combination of approaches—including genetic complementation assays, X-ray crystallography, Langevin dynamics simulations, and small-angle X-ray scattering—we show that the C-terminal region is partially unstructured and has high conformational flexibility. This implies that the C-terminal region acts as a flexible tether linking the polymerase domain to PCNA thereby increasing its local concentration. Such tethering would facilitate the sampling of translesion synthesis polymerases to ensure that the most appropriate one is selected to bypass the lesion.

The translesion DNA polymerases Pol ζ and Rev1 are activated independently of PCNA ubiquitination upon UV radiation in mutants of DNA polymerase δ.

Replicative DNA polymerases cannot insert efficiently nucleotides at sites of base lesions. This function is taken over by specialized translesion DNA synthesis (TLS) polymerases to allow DNA replication completion in the presence of DNA damage. In eukaryotes, Rad6- and Rad18-mediated PCNA ubiquitination at lysine 164 promotes recruitment of TLS polymerases, allowing cells to efficiently cope with DNA damage. However, several studies showed that TLS polymerases can be recruited also in the absence of PCNA ubiquitination. We hypothesized that the stability of the interactions between DNA polymerase δ (Pol δ) subunits and/or between Pol δ and PCNA at the primer/template junction is a crucial factor to determine the requirement of PCNA ubiquitination. To test this hypothesis, we used a structural mutant of Pol δ in which the interaction between Pol3 and Pol31 is inhibited. We found that in yeast, rad18Δ-associated UV hypersensitivity is suppressed by pol3-ct, a mutant allele of the POL3 gene that encodes the catalytic subunit of replicative Pol δ. pol3-ct suppressor effect was specifically dependent on the Rev1 and Pol ζ TLS polymerases. This result strongly suggests that TLS polymerases could rely much less on PCNA ubiquitination when Pol δ interaction with PCNA is partially compromised by mutations. In agreement with this model, we found that the pol3-FI allele suppressed rad18Δ-associated UV sensitivity as observed for pol3-ct. This POL3 allele carries mutations within a putative PCNA Interacting Peptide (PIP) motif. We then provided molecular and genetic evidence that this motif could contribute to Pol δ-PCNA interaction indirectly, although it is not a bona fide PIP. Overall, our results suggest that the primary role of PCNA ubiquitination is to allow TLS polymerases to outcompete Pol δ for PCNA access upon DNA damage.

Melanoma cells replicate through chemotherapy by reducing levels of key homologous recombination protein RAD51 and increasing expression of translesion synthesis DNA polymerase \u03b6

BackgroundThe global incidence of melanoma has been increasing faster than any other form of cancer. New therapies offer exciting prospects for improved survival, but the development of resistance is a major problem and there remains a need for additional effective melanoma therapy. Platinum compounds, such as cisplatin, are the most effective chemotherapeutics for a number of major cancers, but are ineffective on metastatic melanoma. They cause monofunctional adducts and intrastrand crosslinks that are repaired by nucleotide excision repair, as well as the more toxic interstrand crosslinks that are repaired by a combination of nuclease activity and homologous recombination.MethodsWe investigated the mechanism of melanoma resistance to cisplatin using a panel of melanoma and control cell lines. Cisplatin-induced changes in levels of the key homologous recombination protein RAD51 and compensatory changes in translesion synthesis DNA polymerases were identified by western blotting and qRT-PCR. Flow cytometry, immunofluorescence and western blotting were used to compare the cell cycle and DNA damage response and the induction of apoptosis in cisplatin-treated melanoma and control cells. Ectopic expression of a tagged form of RAD51 and siRNA knockdown of translesion synthesis DNA polymerase zeta were used to investigate the mechanism that allowed cisplatin-treated melanoma cells to continue to replicate.ResultsWe have identified and characterised a novel DNA damage response mechanism in melanoma. Instead of increasing levels of RAD51 on encountering cisplatin-induced interstrand crosslinks during replication, melanoma cells shut down RAD51 synthesis and instead boost levels of translesion synthesis DNA polymerase zeta to allow replication to proceed. This response also resulted in synthetic lethality to the PARP inhibitor olaparib.ConclusionsThis unusual DNA damage response may be a more appropriate strategy for an aggressive and rapidly growing tumour like melanoma that enables it to better survive chemotherapy, but also results in increased sensitivity of cultured melanoma cells to the PARP inhibitor olaparib.

Translesion synthesis DNA polymerases promote error-free replication through the minor-groove DNA adduct 3-deaza-3-methyladenine

N3-Methyladenine (3-MeA) is formed in DNA by reaction with S-adenosylmethionine, the reactive methyl donor, and by reaction with alkylating agents. 3-MeA protrudes into the DNA minor groove and strongly blocks synthesis by replicative DNA polymerases (Pols). However, the mechanisms for replicating through this lesion in human cells remain unidentified. Here we analyzed the roles of translesion synthesis (TLS) Pols in the replication of 3-MeA-damaged DNA in human cells. Because 3-MeA has a short half-life in vitro, we used the stable 3-deaza analog, 3-deaza-3-methyladenine (3-dMeA), which blocks the DNA minor groove similarly to 3-MeA. We found that replication through the 3-dMeA adduct is mediated via three different pathways, dependent upon Polι/Polκ, Polθ, and Polζ. As inferred from biochemical studies, in the Polι/Polκ pathway, Polι inserts a nucleotide (nt) opposite 3-dMeA and Polκ extends synthesis from the inserted nt. In the Polθ pathway, Polθ carries out both the insertion and extension steps of TLS opposite 3-dMeA, and in the Polζ pathway, Polζ extends synthesis following nt insertion by an as yet unidentified Pol. Steady-state kinetic analyses indicated that Polι and Polθ insert the correct nt T opposite 3-dMeA with a much reduced catalytic efficiency and that both Pols exhibit a high propensity for inserting a wrong nt opposite this adduct. However, despite their low fidelity of synthesis opposite 3-dMeA, TLS opposite this lesion replicates DNA in a highly error-free manner in human cells. We discuss the implications of these observations for TLS mechanisms in human cells.

Mechanism of error-free replication across benzo[a]pyrene stereoisomers by Rev1 DNA polymerase

Benzo[a]pyrene (BP) is a carcinogen in cigarette smoke which, after metabolic activation, can react with the exocyclic N2 amino group of guanine to generate four stereoisomeric BP-N2-dG adducts. Rev1 is unique among translesion synthesis DNA polymerases in employing a protein-template-directed mechanism of DNA synthesis opposite undamaged and damaged guanine. Here we report high-resolution structures of yeast Rev1 with three BP-N2-dG adducts, namely the 10S (+)-trans-BP-N2-dG, 10R (+)-cis-BP-N2-dG, and 10S ( − )-cis-BP-N2-dG. Surprisingly, in all three structures, the bulky and hydrophobic BP pyrenyl residue is entirely solvent-exposed in the major groove of the DNA. This is very different from the adduct alignments hitherto observed in free or protein-bound DNA. All complexes are well poised for dCTP insertion. Our structures provide a view of cis-BP-N2-dG adducts in a DNA polymerase active site, and offer a basis for understanding error-free replication of the BP-derived stereoisomeric guanine adducts.

Translesion synthesis DNA polymerase \u03b7 exhibits a specific RNA extension activity and a transcription-associated function

Polymerase eta (Polη) is a low fidelity translesion synthesis DNA polymerase that rescues damage-stalled replication by inserting deoxy-ribonucleotides opposite DNA damage sites resulting in error-free or mutagenic damage bypass. In this study we identify a new specific RNA extension activity of Polη of Saccharomyces cerevisiae. We show that Polη is able to extend RNA primers in the presence of ribonucleotides (rNTPs), and that these reactions are an order of magnitude more efficient than the misinsertion of rNTPs into DNA. Moreover, during RNA extension Polη performs error-free bypass of the 8-oxoguanine and thymine dimer DNA lesions, though with a 103 and 102–fold lower efficiency, respectively, than it synthesizes opposite undamaged nucleotides. Furthermore, in vivo experiments demonstrate that the transcription of several genes is affected by the lack of Polη, and that Polη is enriched over actively transcribed regions. Moreover, inactivation of its polymerase activity causes similar transcription inhibition as the absence of Polη. In summary, these results suggest that the new RNA synthetic activity of Polη can have in vivo relevance.