Mutagenic Translesion Synthesis Research Articles

The SOS response-associated peptidase (SRAP) domain of YedK catalyzes ring opening of abasic sites and reversal of its DNA–protein cross-link

Apurinic/apyrimidinic (AP, or abasic) sites in DNA are one of the most common forms of DNA damage. AP sites are reactive and form cross-links to both proteins and DNA, are prone to strand breakage, and inhibit DNA replication and transcription. The replication-associated AP site repair protein HMCES protects cells from strand breaks, inhibits mutagenic translesion synthesis, and participates in repair of interstrand DNA cross-links derived from AP sites by forming a stable thiazolidine DNA–protein cross-link (DPC) to AP sites in single-stranded DNA (ssDNA). Despite the importance of HMCES to genome maintenance and the evolutionary conservation of its catalytic SRAP (SOS Response Associated Peptidase) domain, the enzymatic mechanisms of DPC formation and resolution are unknown. Using the bacterial homolog YedK, we show that the SRAP domain catalyzes conversion of the AP site to its reactive, ring-opened aldehyde form, and we provide structural evidence for the Schiff base intermediate that forms prior to the more stable thiazolidine. We also report two new activities, whereby SRAP reacts with polyunsaturated aldehydes at DNA 3′-ends generated by bifunctional DNA glycosylases and catalyzes direct reversal of the DPC to regenerate the AP site, the latter of which we observe in both YedK and HMCES-SRAP proteins. Taken together, this work provides insights into possible mechanisms by which HMCES DPCs are resolved in cells.

Disrupting the MAD2L2-Rev1 Complex Enhances Cell Death upon DNA Damage.

DNA-damaging chemotherapy agents such as cisplatin have been the first line of treatment for cancer for decades. While chemotherapy can be very effective, its long-term success is often reduced by intrinsic and acquired drug resistance, accompanied by chemotherapy-resistant secondary malignancies. Although the mechanisms causing drug resistance are quite distinct, they are directly connected to mutagenic translesion synthesis (TLS). The TLS pathway promotes DNA damage tolerance by supporting both replication opposite to a lesion and inaccurate single-strand gap filling. Interestingly, inhibiting TLS reduces both cisplatin resistance and secondary tumor formation. Therefore, TLS targeting is a promising strategy for improving chemotherapy. MAD2L2 (i.e., Rev7) is a central protein in TLS. It is an essential component of the TLS polymerase zeta (ζ), and it forms a regulatory complex with Rev1 polymerase. Here we present the discovery of two small molecules, c#2 and c#3, that directly bind both in vitro and in vivo to MAD2L2 and influence its activity. Both molecules sensitize lung cancer cell lines to cisplatin, disrupt the formation of the MAD2L2-Rev1 complex and increase DNA damage, hence underlining their potential as lead compounds for developing novel TLS inhibitors for improving chemotherapy treatments.

REV1 Inhibition Enhances Radioresistance and Autophagy.

Simple SummaryCancer resistance to therapy continues to be the biggest challenge in treating patients. Targeting the mutagenic translesion synthesis (TLS) polymerase REV1 was previously shown to sensitize cancer cells to chemotherapy. In this study, we tested the ability of REV1 inhibitors to radiation therapy and observed a lack of radiosensitization. In addition, we observed REV1 inhibition to trigger an autophagy stress response. Because reduction of REV1 triggered autophagy and failed to radiosensitize cells, we hypothesize REV1 expression dynamics might link cancer cell response to radiation treatment through the potential induction of autophagy.Cancer therapy resistance is a persistent clinical challenge. Recently, inhibition of the mutagenic translesion synthesis (TLS) protein REV1 was shown to enhance tumor cell response to chemotherapy by triggering senescence hallmarks. These observations suggest REV1’s important role in determining cancer cell response to chemotherapy. Whether REV1 inhibition would similarly sensitize cancer cells to radiation treatment is unknown. This study reports a lack of radiosensitization in response to REV1 inhibition by small molecule inhibitors in ionizing radiation-exposed cancer cells. Instead, REV1 inhibition unexpectedly triggers autophagy, which is a known biomarker of radioresistance. We report a possible role of the REV1 TLS protein in determining cancer treatment outcomes depending upon the type of DNA damage inflicted. Furthermore, we discover that REV1 inhibition directly triggers autophagy, an uncharacterized REV1 phenotype, with a significant bearing on cancer treatment regimens.

Biochemical and photochemical mechanisms that produce different UV-induced mutation spectra.

Although UV-induced mutagenesis has been studied extensively, the precise mechanisms that convert UV-induced DNA damage into mutations remain elusive. One well-studied mechanism involves DNA polymerase (Pol) η and ζ, which produces C > T transitions during translesion synthesis (TLS) across pyrimidine dimers. We previously proposed another biochemical mechanism that involves multiple UV-irradiations with incubation in the dark in between. The incubation facilitates spontaneous deamination of cytosine in a pyrimidine dimer, and the subsequent UV irradiation induces photolyase-independent (direct) photoreversal that converts cytosine into monomeric uracil residue. In this paper, we first demonstrate that natural sunlight can induce both mutational processes in vitro. The direct photoreversal was also reproduced by monochromatic UVB at 300 nm. We also demonstrate that post-irradiation incubation in the dark is required for both mutational processes, suggesting that cytosine deamination is required for both the Pol η/ζ-dependent and the photoreversal-dependent mechanisms. Another Y-family polymerase Pol ι also mediated a mutagenic TLS on UV-damaged templates when combined with Pol ζ. The Pol ι-dependent mutations were largely independent of post-irradiation incubation, indicating that cytosine deamination was not essential for this mutational process. Sunlight-exposure also induced C > A transversions which were likely caused by oxidation of guanine residues. Finally, we constructed in vitro mutation spectra in a comparable format to cancer mutation signatures. While both Pol η-dependent and photoreversal-dependent spectra showed high similarities to a cancer signature (SBS7a), Pol ι-dependent mutation spectrum has distinct T > A/C substitutions, which are found in another cancer signature (SBS7d). The Pol ι-dependent T > A/C substitutions were resistant to T4 pyrimidine dimer glycosylase-treatment, suggesting that this mutational process is independent of cis-syn pyrimidine dimers. An updated model about multiple mechanisms of UV-induced mutagenesis is discussed.

RPA2 winged-helix domain facilitates UNG-mediated removal of uracil from ssDNA; implications for repair of mutagenic uracil at the replication fork

Uracil occurs at replication forks via misincorporation of deoxyuridine monophosphate (dUMP) or via deamination of existing cytosines, which occurs 2–3 orders of magnitude faster in ssDNA than in dsDNA and is 100% miscoding. Tethering of UNG2 to proliferating cell nuclear antigen (PCNA) allows rapid post-replicative removal of misincorporated uracil, but potential ‘pre-replicative’ removal of deaminated cytosines in ssDNA has been questioned since this could mediate mutagenic translesion synthesis and induction of double-strand breaks. Here, we demonstrate that uracil-DNA glycosylase (UNG), but not SMUG1 efficiently excises uracil from replication protein A (RPA)-coated ssDNA and that this depends on functional interaction between the flexible winged-helix (WH) domain of RPA2 and the N-terminal RPA-binding helix in UNG. This functional interaction is promoted by mono-ubiquitination and diminished by cell-cycle regulated phosphorylations on UNG. Six other human proteins bind the RPA2-WH domain, all of which are involved in DNA repair and replication fork remodelling. Based on this and the recent discovery of the AP site crosslinking protein HMCES, we propose an integrated model in which templated repair of uracil and potentially other mutagenic base lesions in ssDNA at the replication fork, is orchestrated by RPA. The UNG:RPA2-WH interaction may also play a role in adaptive immunity by promoting efficient excision of AID-induced uracils in transcribed immunoglobulin loci.

REV1 inhibitor JH-RE-06 enhances tumor cell response to chemotherapy by triggering senescence hallmarks

REV1/POLζ-dependent mutagenic translesion synthesis (TLS) promotes cell survival after DNA damage but is responsible for most of the resulting mutations. A novel inhibitor of this pathway, JH-RE-06, promotes cisplatin efficacy in cancer cells and mouse xenograft models, but the mechanism underlying this combinatorial effect is not known. We report that, unexpectedly, in two different mouse xenograft models and four human and mouse cell lines we examined in vitro cisplatin/JH-RE-06 treatment does not increase apoptosis. Rather, it increases hallmarks of senescence such as senescence-associated β-galactosidase, increased p21 expression, micronuclei formation, reduced Lamin B1, and increased expression of the immune regulators IL6 and IL8 followed by cell death. Moreover, although p-γ-H2AX foci formation was elevated and ATR expression was low in single agent cisplatin-treated cells, the opposite was true in cells treated with cisplatin/JH-RE-06. These observations suggest that targeting REV1 with JH-RE-06 profoundly affects the nature of the persistent genomic damage after cisplatin treatment and also the resulting physiological responses. These data highlight the potential of REV1/POLζ inhibitors to alter the biological response to DNA-damaging chemotherapy and enhance the efficacy of chemotherapy.

A stapled POL κ peptide targets REV1 to inhibit mutagenic translesion synthesis.

Stapled α-helical RIR (Rev1-interacting region) peptides of DNA POL κ bind more effectively to the RIR-interface of the C-terminal recruitment domain of the translesion synthesis DNA polymerase Rev1 than unstapled peptide. The tightest-binding stapled peptide translocates into cells and enhances the cytotoxicity of DNA damaging agents while reducing mutagenesis. Drugs with these characteristics could potentially serve as adjuvants to improve chemotherapy and reduce acquired resistance by inhibiting Rev1-dependent mutagenic translesion synthesis.

Translesion synthesis genes POLI and REV1 in breast tumors and response to neoadjuvant chemotherapy.

e12642 Background: Translesion synthesis (TLS) of DNA by specialized polymerases (POLs) and REV1, a scaffold protein that recruits other TLS DNA POLs for nascent strand extension, experimentally enable tumor cells to replicate through DNA damage. TLS POLs are hypothesized to contribute to treatment resistance. Methods: We conducted a discovery gene expression study using NanoString technology with a bespoke codeset enriched for DNA damage response genes and RNA from 12 TN, 10 HER2+ and 29 ER+/HER2- breast cancer patients before neoadjuvant chemotherapy (NACT) and when available, in the matching residual tumor. Eleven patients achieved a pathologic complete response (pCR), 12 had < 1 cm residual disease (partial responders) and 28 patients had > 1cm residual disease (non-responders). RNA counts were normalized and log-transformed for differential expression analyses corrected for false discovery. Results: DNA polymerase Iota ( POLI) was the top overexpressed gene in the pretreatment biopsy of non-responders independent of tumor subtype (p corrected = 0.0002). Expression of REV1 was also significantly higher in non-responders and positively correlated with POLI (r = 0.577, p < 0.0001). Despite a small sample size, pretreatment expression levels of POLI and REV1 were significantly higher in ER+ (n = 38) than ER- (n = 13) tumors (p = 0.0006). When restricted to ER+ tumors, POL1 and REV1 expression levels were significantly higher in pretreatment biopsy of non-responders (n = 25) than partial and complete responders (n = 13). For ER+ patients, response by median POL1/REV1 expression levels are shown in Table. Pre/post treatment POL1 and REV1 expression did not change in patients with residual disease. Conclusions: In discovery, two genes involved in TLS of DNA (POLI and REV1) were found overexpressed in pretreatment biopsies from breast cancers poorly responsive to NACT, independent of tumor subtype. Overexpression was more common among ER+ tumors, which exhibit a high inherent resistance to chemotherapy. If confirmed, expressed levels of POLI and REV1 may identify a subgroup of breast cancers resistant to NACT for which targeted inhibition of mutagenic TLS may be beneficial. [Table: see text]

Translesion Synthesis in Plants: Ultraviolet Resistance and Beyond.

Plant genomes sustain various forms of DNA damage that stall replication forks. Translesion synthesis (TLS) is one of the pathways to overcome stalled replication in which specific polymerases (TLS polymerase) perform bypass synthesis across DNA damage. This article gives a brief overview of plant TLS polymerases. In Arabidopsis, DNA polymerase (Pol) ζ, η, κ, θ, and λ and Reversionless1 (Rev1) are shown to be involved in the TLS. For example, AtPolη bypasses ultraviolet (UV)-induced cyclobutane pyrimidine dimers in vitro. Disruption of AtPolζ or AtPolη increases root stem cell death after UV irradiation. These results suggest that AtPolζ and ATPolη bypass UV-induced damage, prevent replication arrest, and allow damaged cells to survive and grow. In general, TLS polymerases have low fidelity and often induce mutations. Accordingly, disruption of AtPolζ or AtRev1 reduces somatic mutation frequency, whereas disruption of AtPolη elevates it, suggesting that plants have both mutagenic and less mutagenic TLS activities. The stalled replication fork can be resolved by a strand switch pathway involving a DNA helicase Rad5. Disruption of both AtPolζ and AtRAD5a shows synergistic or additive effects in the sensitivity to DNA-damaging agents. Moreover, AtPolζ or AtRev1 disruption elevates homologous recombination frequencies in somatic tissues. These results suggest that the Rad5-dependent pathway and TLS are parallel. Plants grown in the presence of heat shock protein 90 (HSP90) inhibitor showed lower mutation frequencies, suggesting that HSP90 regulates mutagenic TLS in plants. Hypersensitivities of TLS-deficient plants to γ-ray and/or crosslink damage suggest that plant TLS polymerases have multiple roles, as reported in other organisms.

ATR kinase inhibition sensitizes quiescent human cells to the lethal effects of cisplatin but increases mutagenesis

The ATR protein kinase is known to protect cells from DNA damage induced during the replicative phase of the cell cycle. Small molecule ATR kinase inhibitors have therefore been developed to improve the effectiveness of DNA damage-based chemotherapy regimens aimed at killing rapidly proliferating tumor cells. However, whether ATR functions in a similar manner in non-replicating cells has not been examined and is important considering the fact that most cells in the body, including cancer stem cells in solid tumors, normally reside in either a quiescent or differentiated non-replicating state. Using cultured human cell lines maintained in a quiescent or slowly growing state in vitro, ATR was found to be activated following treatment with the common anti-cancer drug cisplatin in a manner dependent on the nucleotide excision repair (NER) system. Moreover, treatment with the ATR kinase inhibitors VE-821 and AZD6738 enhanced quiescent cell killing and apoptotic signaling induced by cisplatin. However, ATR kinase inhibition in quiescent cells treated with a low concentration of cisplatin also elevated the level of mutagenesis at the hypoxanthine phosphoribosyltransferase locus and resulted in increased levels of PCNA mono-ubiquitination. These results suggest that the excision gaps generated by NER may require a greater utilization of potentially mutagenic translesion synthesis polymerases in the absence of ATR kinase function. Thus, though ATR kinase inhibitors can aid in the killing of cisplatin-treated quiescent cells, such treatments may also result in a greater reliance on alternative mutagenic DNA polymerases to complete the repair of cisplatin-DNA adducts.

A Small Molecule Targeting Mutagenic Translesion Synthesis Improves Chemotherapy

Intrinsic and acquired drug resistance and induction ofsecondary malignancies limit successful chemotherapy. Because mutagenic translesion synthesis (TLS) contributes to chemoresistance as well as treatment-induced mutations, targeting TLS is an attractive avenue for improving chemotherapeutics. However, development of small molecules with high specificity and invivo efficacy for mutagenic TLS has been challenging. Here, we report the discovery of a small-moleculeinhibitor, JH-RE-06, that disrupts mutagenic TLS by preventing recruitment of mutagenic POL ζ. Remarkably, JH-RE-06 targets a nearly featureless surface of REV1 that interacts with the REV7 subunit of POL ζ. Binding of JH-RE-06 induces REV1 dimerization, which blocks the REV1-REV7 interaction and POL ζ recruitment. JH-RE-06 inhibits mutagenic TLS and enhances cisplatin-induced toxicity in cultured human and mouse cell lines. Co-administration of JH-RE-06 with cisplatin suppresses the growth of xenograft humanmelanomas in mice, establishing a framework for developing TLS inhibitors as a novel class of chemotherapy adjuvants.

Abstract 1354: TonEBP mediates PCNA polyubiquitination on DNA damage sites via interaction with SHPRH and USP1

Abstract PCNA ubiquitination is important for DNA damage tolerance in eukaryotic replication. While PCNA monoubiquitination activates a mutagenic translesion synthesis pathway, polyubiquitination activates an error-free template switching for DNA damage bypass. Even though PCNA polyubiquitination is critical for the maintenance of genomic integrity, underlying mechanisms are poorly understood. Here, we report that TonEBP (tonicity-responsive enhancer binding protein) is involved the PCNA polyubiquitination in response to DNA damage. TonEBP sequentially recruited an E3 ubiquitin ligase SHPRH followed by a deubiquitinase USP1 into DNA damaged sites, which showed direct correlation with dynamics of PCNA polyubiquitination. The Rel-homology domain (RHD) of TonEBP, which encircles DNA, was required for the interaction with SHRPH and USP1. Consistent with its importance for DNA damage bypass, knockdown or deletion of RHD in TonEBP increased sensitivity to methyl methanesulfonate. Collectively, TonEBP is a key player in the post-replication DNA repair. Citation Format: Hyunje Kang, Hyun Park, Eun Jin Yoo, Jun Ho Lee, Soo Youn Choi, Whaseon Lee-Kwon, Kyoo-young Lee, Jin-Hoe Hur, Jeong Kon Seo, Kyungjae Myung, Hyug Moo Kwon. TonEBP mediates PCNA polyubiquitination on DNA damage sites via interaction with SHPRH and USP1 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1354.

Replication Fork Slowing and Reversal upon DNA Damage Require PCNA Polyubiquitination and ZRANB3 DNA Translocase Activity

SummaryDNA damage tolerance during eukaryotic replication is orchestrated by PCNA ubiquitination. While monoubiquitination activates mutagenic translesion synthesis, polyubiquitination activates an error-free pathway, elusive in mammals, enabling damage bypass by template switching. Fork reversal is driven in vitro by multiple enzymes, including the DNA translocase ZRANB3, shown to bind polyubiquitinated PCNA. However, whether this interaction promotes fork remodeling and template switching in vivo was unknown. Here we show that damage-induced fork reversal in mammalian cells requires PCNA ubiquitination, UBC13, and K63-linked polyubiquitin chains, previously involved in error-free damage tolerance. Fork reversal in vivo also requires ZRANB3 translocase activity and its interaction with polyubiquitinated PCNA, pinpointing ZRANB3 as a key effector of error-free DNA damage tolerance. Mutations affecting fork reversal also induced unrestrained fork progression and chromosomal breakage, suggesting fork remodeling as a global fork slowing and protection mechanism. Targeting these fork protection systems represents a promising strategy to potentiate cancer chemotherapy.

Editor's Highlight: High-Throughput Functional Genomics Identifies Modulators of TCE Metabolite Genotoxicity and Candidate Susceptibility Genes.

Trichloroethylene (TCE), an industrial chemical and environmental contaminant, is a human carcinogen. Reactive metabolites are implicated in renal carcinogenesis associated with TCE exposure, yet the toxicity mechanisms of these metabolites and their contribution to cancer and other adverse effects remain unclear. We employed an integrated functional genomics approach that combined functional profiling studies in yeast and avian DT40 cell models to provide new insights into the specific mechanisms contributing to toxicity associated with TCE metabolites. Genome-wide profiling studies in yeast identified the error-prone translesion synthesis (TLS) pathway as an import mechanism in response to TCE metabolites. The role of TLS DNA repair was further confirmed by functional profiling in DT40 avian cell lines, but also revealed that TLS and homologous recombination DNA repair likely play competing roles in cellular susceptibility to TCE metabolites in higher eukaryotes. These DNA repair pathways are highly conserved between yeast, DT40, and humans. We propose that in humans, mutagenic TLS is favored over homologous recombination repair in response to TCE metabolites. The results of these studies contribute to the body of evidence supporting a mutagenic mode of action for TCE-induced renal carcinogenesis mediated by reactive metabolites in humans. Our approach illustrates the potential for high-throughput in vitro functional profiling in yeast to elucidate toxicity pathways (molecular initiating events, key events) and candidate susceptibility genes for focused study.

Inhibition of mutagenic translesion synthesis: A possible strategy for improving chemotherapy?

While DNA damaging chemotherapy can be very effective and even curative in the treatment of certain cancers, intrinsic and acquired drug resistance underlies tumor progression and morbidity in many cancer patients. Intrinsic resistance defines a cell state that is inherently tolerant of drug action. This can include the activation of drug efflux pumps or detoxifying processes that effectively reduce intracellular drug concentration [1]. This can also include a change in the recognition or persistence of DNA damage, mediated by an enhanced DNA repair capability, a blunted DNA damage response, or the ability to proliferate in the presence of DNA damage. Conversely, acquired drug resistance represents a mutational or epigenetic process by which a chemosensitive cell develops 1 or more of the characteristics of an intrinsically resistant cancer cell. Thus, the mechanisms underlying intrinsic and acquired drug resistance are quite distinct. One describes a cell state, and the other describes the capability of reaching that cell state. Yet, these processes are very much coupled in the context of mutagenic translesion synthesis (TLS). As discussed throughout this review, mutagenic TLS polymerases underlie 2 important phenotypes in response to genotoxic chemotherapy. First, they allow for the bypass of modified DNA bases during DNA synthesis, allowing proliferation to continue in the presence of chemotherapy. Second, the low fidelity replication performed by TLS polymerases results in the introduction of inappropriate, nonpairing bases across from modified nucleotides. The bypass function of TLS polymerases is particularly relevant to intrinsic drug resistance. Many tumors, including most pancreatic adenocarcinomas, nonsmall cell lung cancers, and aggressive brain tumors, as well as most metastatic malignancies, fail to significantly regress following chemotherapy [2]. In these tumors, TLS activity contributes to a drug resistant state by promoting the tolerance of DNA damage [3–6]. Conversely, the mutational role of TLS polymerases is central to process of acquired drug resistance. Tumor regression and relapse following chemotherapy is almost always accompanied by the development of drug resistant disease. This may not occur at initial relapse, but upon serial cycles of treatment patients generally succumb to tumors that have acquired intrinsically resistant disease. In fact, for certain cancers the overall prognosis is not dictated by the initial response of the tumor to chemotherapy. Rather, the response of the relapsed tumor to therapy is a significantly better determinant of overall survival. For instance, a high error-prone TLS activity translates into greater tumor adaptation to chemotherapy, while a low error-prone TLS activity leaves tumor in a treatment-naive state. This latter state is amenable to continued long-term treatment of tumors that remain response to treatment with the initial therapy. The dual functions of mutagenic TLS polymerases in intrinsic and acquired chemoresistance make these proteins very attractive potential targets for adjuvant therapy. When combined with front-line genotoxic therapy, these TLS inhibitors would be expected to sensitize tumors to chemotherapy while blocking drug-induced mutation. Consequently, while the generation of such inhibitors is complex, their route to the clinic is more apparent. TLS inhibitors could be applied in combination with the standard of care for many malignancies. By effectively increasing the effects of chemotherapy in target cells, these agents may also allow for a reduction in chemotherapy dose regimens. An added benefit of these agents may be a reduction in the rate of secondary chemotherapy-driven malignancies that occur in patients following successful treatment of the primary disease.

Mutations at the Subunit Interface of Yeast Proliferating Cell Nuclear Antigen Reveal a Versatile Regulatory Domain.

Proliferating cell nuclear antigen (PCNA) plays a key role in many cellular processes and due to that it interacts with a plethora of proteins. The main interacting surfaces of Saccharomyces cerevisiae PCNA have been mapped to the interdomain connecting loop and to the carboxy-terminal domain. Here we report that the subunit interface of yeast PCNA also has regulatory roles in the function of several DNA damage response pathways. Using site-directed mutagenesis we engineered mutations at both sides of the interface and investigated the effect of these alleles on DNA damage response. Genetic experiments with strains bearing the mutant alleles revealed that mutagenic translesion synthesis, nucleotide excision repair, and homologous recombination are all regulated through residues at the subunit interface. Moreover, genetic characterization of one of our mutants identifies a new sub-branch of nucleotide excision repair. Based on these results we conclude that residues at the subunit boundary of PCNA are not only important for the formation of the trimer structure of PCNA, but they constitute a regulatory protein domain that mediates different DNA damage response pathways, as well.

Rev1 promotes replication through UV lesions in conjunction with DNA polymerases η, ι, and κ but not DNA polymerase ζ.

Translesion synthesis (TLS) DNA polymerases (Pols) promote replication through DNA lesions; however, little is known about the protein factors that affect their function in human cells. In yeast, Rev1 plays a noncatalytic role as an indispensable component of Polζ, and Polζ together with Rev1 mediates a highly mutagenic mode of TLS. However, how Rev1 functions in TLS and mutagenesis in human cells has remained unclear. Here we determined the role of Rev1 in TLS opposite UV lesions in human and mouse fibroblasts and showed that Rev1 is indispensable for TLS mediated by Polη, Polι, and Polκ but is not required for TLS by Polζ. In contrast to its role in mutagenic TLS in yeast, Rev1 promotes predominantly error-free TLS opposite UV lesions in humans. The identification of Rev1 as an indispensable scaffolding component for Polη, Polι, and Polκ, which function in TLS in highly specialized ways opposite a diverse array of DNA lesions and act in a predominantly error-free manner, implicates a crucial role for Rev1 in the maintenance of genome stability in humans.

DNA polymerases κ and ζ cooperatively perform mutagenic translesion synthesis of the C8-2'-deoxyguanosine adduct of the dietary mutagen IQ in human cells.

The roles of translesion synthesis (TLS) DNA polymerases in bypassing the C8–2′-deoxyguanosine adduct (dG-C8-IQ) formed by 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), a highly mutagenic and carcinogenic heterocyclic amine found in cooked meats, were investigated. Three plasmid vectors containing the dG-C8-IQ adduct at the G1-, G2- or G3-positions of the NarI site (5′-G1G2CG3CC-3′) were replicated in HEK293T cells. Fifty percent of the progeny from the G3 construct were mutants, largely G→T, compared to 18% and 24% from the G1 and G2 constructs, respectively. Mutation frequency (MF) of dG-C8-IQ was reduced by 38–67% upon siRNA knockdown of pol κ, whereas it was increased by 10–24% in pol η knockdown cells. When pol κ and pol ζ were simultaneously knocked down, MF of the G1 and G3 constructs was reduced from 18% and 50%, respectively, to <3%, whereas it was reduced from 24% to <1% in the G2 construct. In vitro TLS using yeast pol ζ showed that it can extend G3*:A pair more efficiently than G3*:C pair, but it is inefficient at nucleotide incorporation opposite dG-C8-IQ. We conclude that pol κ and pol ζ cooperatively carry out the majority of the error-prone TLS of dG-C8-IQ, whereas pol η is involved primarily in its error-free bypass.

DNA polymerase ζ-dependent lesion bypass in Saccharomyces cerevisiae is accompanied by error-prone copying of long stretches of adjacent DNA.

Translesion synthesis (TLS) helps cells to accomplish chromosomal replication in the presence of unrepaired DNA lesions. In eukaryotes, the bypass of most lesions involves a nucleotide insertion opposite the lesion by either a replicative or a specialized DNA polymerase, followed by extension of the resulting distorted primer terminus by DNA polymerase ζ (Polζ). The subsequent events leading to disengagement of the error-prone Polζ from the primer terminus and its replacement with an accurate replicative DNA polymerase remain largely unknown. As a first step toward understanding these events, we aimed to determine the length of DNA stretches synthesized in an error-prone manner during the Polζ-dependent lesion bypass. We developed new in vivo assays to identify the products of mutagenic TLS through a plasmid-borne tetrahydrofuran lesion and a UV-induced chromosomal lesion. We then surveyed the region downstream of the lesion site (in respect to the direction of TLS) for the presence of mutations indicative of an error-prone polymerase activity. The bypass of both lesions was associated with an approximately 300,000-fold increase in the mutation rate in the adjacent DNA segment, in comparison to the mutation rate during normal replication. The hypermutated tract extended 200 bp from the lesion in the plasmid-based assay and as far as 1 kb from the lesion in the chromosome-based assay. The mutation rate in this region was similar to the rate of errors produced by purified Polζ during copying of undamaged DNA in vitro. Further, no mutations downstream of the lesion were observed in rare TLS products recovered from Polζ-deficient cells. This led us to conclude that error-prone Polζ synthesis continues for several hundred nucleotides after the lesion bypass is completed. These results provide insight into the late steps of TLS and show that error-prone TLS tracts span a substantially larger region than previously appreciated.

Roles of mutagenic translesion synthesis in mammalian genome stability, health and disease.

Most spontaneous and DNA damage-induced nucleotide substitutions in eukaryotes depend on translesion synthesis polymerases Rev1 and Pol ζ, the latter consisting of the catalytic subunit Rev3 and the accessory protein Rev7. Here we review the regulation, and the biochemical and cellular functions, of Rev1/Pol ζ-dependent translesion synthesis. These are correlated with phenotypes of mouse models with defects in Rev1, Rev3 or Rev7. The data indicate that Rev1/Pol ζ-mediated translesion synthesis is important for adaptive immunity while playing paradoxical roles in oncogenesis. On the other hand, by enabling the replication of endogenously damaged templates, Rev1/Pol ζ -dependent translesion synthesis protects stem cells, thereby preventing features of ageing. In conclusion, Rev1/Pol ζ-dependent translesion synthesis at DNA helix-distorting nucleotide lesions orchestrates pleiotropic responses that determine organismal fitness and disease.