Translesion Synthesis Research Articles

Zebrafish Polymerase Theta and human Polymerase Theta: orthologues with homologous function.

DNA Polymerase Theta (Pol θ) is a conserved an A-family polymerase that plays an essential role in repairing double strand breaks, through micro-homology end joining, and bypassing DNA lesions, through translesion synthesis, to protect genome integrity. Despite its essential role in DNA repair, Pol θ is inherently error-prone. Recently, key loop regions were identified to play an important role in key functions of Pol θ. Here we present a comparative structure-function study of the polymerase domain of zebrafish and human Pol θ. We show that these two proteins share a large amount of sequence and structural homology. However, we identify differences in the amino acid composition within the key loop areas shown to drive characteristic Pol θ functions. Despite these differences zebrafish Pol θ still displays characteristics identify in human Pol θ, including DNA template extension in the presence of different divalent metals, microhomology-mediated end joining, and translesion synthesis. These results will support future studies looking to gain insight into Pol θ function on the basis of evolutionarily conserved features.

POLQ immunostaining behaves as a prognostic factor for pancreatic carcinoma.

DNA polymerase theta (POLQ) is a translesion synthesis polymerase essential for the repair of double strand breaks by the error-prone TMEJ (Theta Mediated End Joining) pathway. Although POLQ participates in maintaining genome stability, several studies have shown that its overexpression correlates with cancer progression and poor prognosis. Due to the fact that its role as a biomarker in pancreatic cancer remains unexplored, we aimed to study the usefulness of POLQ H-score as a prognostic factor in a pancreatic cancer patient cohort. We evaluated POLQ gene expression using a web-based tool to deliver gene expression profiling and interactive analyses based on TCGA and GTEx (GEPIA) and we examined the POLQ immunostaining in 152 biliopancreatic cancer surgical specimens using tissue microarrays. Association with survival was evaluated by Kaplan Meier curves and uni-multivariate Cox regression. GEPIA analysis showed statistical differences according to POLQ mRNA levels in Disease Free Survival (DFS) (log rank 0.023, HR 2.8, p=0.029) and Overall Survival (OS) (log rank 0.011, HR 3.1, p=0.016). For immunohistochemistry (IHC) evaluation, POLQ H-score was calculated, and showed statistical differences for OS in Kaplan Meier curves (log rank 0.001) and uni-multivariate analysis (HR 2.27; 95% CI 1.24-4.15, p=0.008). Our results indicate that POLQ is an independent prognostic factor in pancreatic cancer when analyzed by immunostaining, which is in agreement with the results shown by the POLQ gene expression analysis (GEPIA).

DNA polymerase ζ has robust reverse transcriptase activity relative to other cellular DNA polymerases.

Cell biology and genetic studies have demonstrated that DNA double strand break (DSB) repair can be performed using an RNA transcript that spans the site of the DNA break as a template for repair. This type of DSB repair requires a reverse transcriptase to convert an RNA sequence into DNA to facilitate repair of the break, rather than copying from a DNA template as in canonical DSB repair. Translesion synthesis (TLS) DNA polymerases (Pol) are often more promiscuous than DNA Pols, raising the notion that reverse transcription could be performed by a TLS Pol. Indeed, several studies have demonstrated that human Pol η has reverse transcriptase activity, while others have suggested that the yeast TLS Pol ζ is involved. Here, we purify all seven known nuclear DNA Pols of Saccharomyces cerevisiae and compare their reverse transcriptase activities. The comparison shows that Pol ζ far surpasses Pol η and all other DNA Pols in reverse transcriptase activity. We find that Pol ζ reverse transcriptase activity is not affected by RPA or RFC/PCNA and acts distributively to make DNA complementary to an RNA template strand. Consistent with prior S. cerevisiae studies performed in vivo, we propose that Pol ζ is the major DNA Pol that functions in the RNA templated DSB repair pathway.

DNA Replication across α-l-(3'-2')-Threofuranosyl Nucleotides Mediated by Human DNA Polymerase η.

α-l-(3'-2')-Threofuranosyl nucleic acid (TNA) pairs with itself, cross-pairs with DNA and RNA, and shows promise as a tool in synthetic genetics, diagnostics, and oligonucleotide therapeutics. We studied in vitro primer insertion and extension reactions catalyzed by human trans-lesion synthesis (TLS) DNA polymerase η (hPol η) opposite a TNA-modified template strand without and in combination with O4-alkyl thymine lesions. Across TNA-T (tT), hPol η inserted mostly dAMP and dGMP, dTMP and dCMP with lower efficiencies, followed by extension of the primer to a full-length product. hPol η inserted dAMP opposite O4-methyl and -ethyl analogs of tT, albeit with reduced efficiencies relative to tT. Crystal structures of ternary hPol η complexes with template tT and O4-methyl tT at the insertion and extension stages demonstrated that the shorter backbone and different connectivity of TNA compared to DNA (3' → 2' versus 5' → 3', respectively) result in local differences in sugar orientations, adjacent phosphate spacings, and directions of glycosidic bonds. The 3'-OH of the primer's terminal thymine was positioned at 3.4 Å on average from the α-phosphate of the incoming dNTP, consistent with insertion opposite and extension past the TNA residue by hPol η. Conversely, the crystal structure of a ternary hPol η·DNA·tTTP complex revealed that the primer's terminal 3'-OH was too distant from the tTTP α-phosphate, consistent with the inability of the polymerase to incorporate TNA. Overall, our study provides a better understanding of the tolerance of a TLS DNA polymerase vis-à-vis unnatural nucleotides in the template and as the incoming nucleoside triphosphate.

Probing hot spots of protein-protein interactions mediated by the safety-belt region of REV7.

REV7 is a HORMA (Hop1, Rev7, Mad2) family adaptor protein best known as an accessory subunit of the translesion synthesis (TLS) DNA polymerase ζ (Polζ). In this role, REV7 binds REV3, the catalytic subunit of Polζ, by locking REV7-binding motifs (RBMs) in REV3 underneath the REV7 safety-belt loop. The same mechanism is used by REV7 to interact with RBMs from other proteins in DNA damage response (DDR) and mitosis. Because of the importance of REV7 for TLS and other DDR pathways, targeting REV7:RBM protein-protein interactions (PPIs) with small molecules has emerged as a strategy to enhance cancer response to genotoxic chemotherapy. To identify druggable pockets at the REV7:RBM interface, we performed computational analyses of REV7 complexed with several RBM partners. The contributions of different interface regions to REV7:RBM stabilization were corroborated experimentally. These studies provide insights into key intermolecular interactions and establish targetable regions of REV7 for the design of REV7:RBM PPI inhibitors.

DNA polymerase ζ is a robust reverse transcriptase.

Cell biology and genetic studies have demonstrated that DNA double strand break (DSB) repair can be performed using an RNA transcript that spans the site of the DNA break as a template for repair. This type of DSB repair requires a reverse transcriptase to convert an RNA sequence into DNA to facilitate repair of the break, rather than copying from a DNA template as in canonical DSB repair. Translesion synthesis (TLS) DNA polymerases (Pol) are often more promiscuous than DNA Pols, raising the notion that reverse transcription could be performed by a TLS Pol. Indeed, several studies have demonstrated that human Pol η has reverse transcriptase activity, while others have suggested that the yeast TLS Pol ζ is involved. Here, we purify all seven known nuclear DNA Pols of Saccharomyces cerevisiae and compare their reverse transcriptase activities. The comparison shows that Pol ζ far surpasses Pol η and all other DNA Pols in reverse transcriptase activity. We find that Pol ζ reverse transcriptase activity is not affected by RPA or RFC/PCNA and acts distributively to make DNA complementary to an RNA template strand. Consistent with prior S. cerevisiae studies performed in vivo, we propose that Pol ζ is the major DNA Pol that functions in the RNA templated DSB repair pathway.

Canonical and Non-Canonical Roles of Human DNA Polymerase η.

DNA damage tolerance pathways that allow for the completion of replication following fork arrest are critical in maintaining genome stability during cell division. The main DNA damage tolerance pathways include strand switching, replication fork reversal and translesion synthesis (TLS). The TLS pathway is mediated by specialised DNA polymerases that can accommodate altered DNA structures during DNA synthesis, and are important in allowing replication to proceed after fork arrest, preventing fork collapse that can generate more deleterious double-strand breaks in the genome. TLS may occur directly at the fork, or at gaps remaining behind the fork, in the process of post-replication repair. Inactivating mutations in the human POLH gene encoding the Y-family DNA polymerase Pol η causes the skin cancer-prone genetic disease xeroderma pigmentosum variant (XPV). Pol η also contributes to chemoresistance during cancer treatment by bypassing DNA lesions induced by anti-cancer drugs including cisplatin. We review the current understanding of the canonical role of Pol η in translesion synthesis following replication arrest, as well as a number of emerging non-canonical roles of the protein in other aspects of DNA metabolism.

Cell type specific suppression of hyper-recombination by human RAD18 is linked to PCNA K164 ubiquitination.

Homologous recombination (HR) and translesion synthesis (TLS) promote gap-filling DNA synthesis to complete genome replication. One factor involved in both pathways is RAD18, an E3 ubiquitin ligase. Although RAD18's role in promoting TLS through the ubiquitination of PCNA at lysine 164 (K164) is well established, its requirement for HR-based mechanisms is currently less clear. To assess this, we inactivated RAD18 in three human cell lines. Our analyses found that loss of RAD18 in HCT116, but neither hTERT RPE-1 nor DLD1 cell lines, resulted in elevated sister chromatid exchange, gene conversion, and gene targeting, i.e . HCT116 mutants were hyper-recombinogenic (hyper-rec). Loss of RAD18 also impaired TLS activity in HCT116 cells, but unexpectedly, did not reduce clonogenic survival. Interestingly, these phenotypes appear linked to PCNA K164 ubiquitination, as HCT116 PCNA K164R/+ mutants were also hyper-rec and showed reduced TLS activity, consistent with previous studies in rad18 -/- or pcna K164R avian DT40 mutant cells. Importantly, knockdown of UBC9 to prevent PCNA K164 SUMOylation did not affect hyper-recombination, strengthening the link between increased recombination and RAD18-catalyzed PCNA K164 ubiquitination, but not K164 SUMOylation. Taken together, these data suggest that the roles of human RAD18 in directing distinct gap-filling DNA synthesis pathways varies depending on cell type and that these functions are linked to PCNA ubiquitination.

Tolerating DNA damage by repriming: Gap filling in the spotlight

Timely and accurate DNA replication is critical for safeguarding genome integrity and ensuring cell viability. Yet, this process is challenged by DNA damage blocking the progression of the replication machinery. To counteract replication fork stalling, evolutionary conserved DNA damage tolerance (DDT) mechanisms promote DNA damage bypass and fork movement. One of these mechanisms involves “skipping” DNA damage through repriming downstream of the lesion, leaving single-stranded DNA (ssDNA) gaps behind the advancing forks (also known as post-replicative gaps). In vertebrates, repriming in damaged leading templates is proposed to be mainly promoted by the primase and polymerase PRIMPOL. In this review, we discuss recent advances towards our understanding of the physiological and pathological conditions leading to repriming activation in human models, revealing a regulatory network of PRIMPOL activity. Upon repriming by PRIMPOL, post-replicative gaps formed can be filled-in by the DDT mechanisms translesion synthesis and template switching. We discuss novel findings on how these mechanisms are regulated and coordinated in time to promote gap filling. Finally, we discuss how defective gap filling and aberrant gap expansion by nucleases underlie the cytotoxicity associated with post-replicative gap accumulation. Our increasing knowledge of this repriming mechanism – from gap formation to gap filling – is revealing that targeting the last step of this pathway is a promising approach to exploit post-replicative gaps in anti-cancer therapeutic strategies.

DNA mismatch repair controls the mutagenicity of Polymerase ζ-dependent translesion synthesis at methylated guanines

By replicating damaged nucleotides, error-prone DNA translesion synthesis (TLS) enables the completion of replication, albeit at the expense of fidelity. TLS of helix-distorting DNA lesions, that usually have reduced capacity of basepairing, comprises insertion opposite the lesion followed by extension, the latter in particular by polymerase ζ (Pol ζ). However, little is known about involvement of Pol ζ in TLS of non- or poorly-distorting, but miscoding, lesions such as O6-methyldeoxyguanosine (O6-medG). Using purified Pol ζ we describe that the enzyme can misincorporate thymidine opposite O6-medG and efficiently extend from terminal mismatches, suggesting its involvement in the mutagenicity of O6-medG. Surprisingly, O6-medG lesions induced by the methylating agent N-methyl-N’-nitro-N-nitrosoguanidine (MNNG) appeared more, rather than less, mutagenic in Pol ζ-deficient mouse embryonic fibroblasts (MEFs) than in wild type MEFs. This suggested that in vivo Pol ζ participates in non-mutagenic TLS of O6-medG. However, we found that the Pol ζ-dependent misinsertions at O6-medG lesions are efficiently corrected by DNA mismatch repair (MMR), which masks the error-proneness of Pol ζ. We also found that the MNNG-induced mutational signature is determined by the adduct spectrum, and modulated by MMR. The signature mimicked single base substitution signature 11 in the catalogue of somatic mutations in cancer, associated with treatment with the methylating drug temozolomide. Our results unravel the individual roles of the major contributors to methylating drug-induced mutagenesis. Moreover, these results warrant caution as to the classification of TLS as mutagenic or error-free based on in vitro data or on the analysis of mutations induced in MMR-proficient cells.

An altered cell-specific subcellular distribution of translesion synthesis DNA polymerase kappa (POLK) in aging neurons.

Genomic stability is critical for cellular function, however, in the central nervous system highly metabolically active differentiated neurons are challenged to maintain their genome over the organismal lifespan without replication. DNA damage in neurons increases with chronological age and accelerates in neurodegenerative disorders, resulting in cellular and systemic dysregulation. Distinct DNA damage response strategies have evolved with a host of polymerases. The Y-family translesion synthesis (TLS) polymerases are well known for bypassing and repairing damaged DNA in dividing cells. However, their expression, dynamics, and role if any, in enduring postmitotic differentiated neurons of the brain are completely unknown. We show through systematic longitudinal studies for the first time that DNA polymerase kappa (POLK), a member of the Y-family polymerases, is highly expressed in neurons. With chronological age, there is a progressive and significant reduction of nuclear POLK with a concomitant accumulation in the cytoplasm that is predictive of brain tissue age. The reduction of nuclear POLK in old brains is congruent with an increase in DNA damage markers. The nuclear POLK colocalizes with damaged sites and DNA repair proteins. The cytoplasmic POLK accumulates with stress granules and endo/lysosomal markers. Nuclear POLK expression is significantly higher in GABAergic interneurons compared to excitatory pyramidal neurons and lowest in non-neurons, possibly reflective of the inherent biological differences such as firing rates and neuronal activity. Interneurons associated with microglia have significantly higher levels of cytoplasmic POLK in old age. Finally, we show that neuronal activity itself can lead to an increase in nuclear POLK levels and a reduction of the cytoplasmic fraction. Our findings open a new avenue in understanding how different classes of postmitotic neurons deploy TLS polymerase(s) to maintain their genomic integrity over time, which will help design strategies for longevity, healthspan, and prevention of neurodegeneration.

"Bridging the DNA divide": Understanding the interplay between replication- gaps and homologous recombination proteins RAD51 and BRCA1/2

A key but often neglected component of genomic instability is the emergence of single-stranded DNA (ssDNA) gaps during DNA replication in the absence of functional homologous recombination (HR) proteins, such as RAD51 and BRCA1/2. Research in prokaryotes has shed light on the dual role of RAD51's bacterial ortholog, RecA, in HR and the protection of replication forks, emphasizing its essential role in preventing the formation of ssDNA gaps, which is vital for cellular viability. This phenomenon was corroborated in eukaryotic cells deficient in HR, where the formation of ssDNA gaps within newly synthesized DNA and their subsequent processing by the MRE11 nuclease were observed. Without functional HR proteins, cells employ alternative ssDNA gap-filling mechanisms to ensure survival, though this compensatory response can compromise genomic stability. A notable example is the involvement of the translesion synthesis (TLS) polymerase POLζ, along with the repair protein POLθ, in the suppression of replicative ssDNA gaps. Persistent ssDNA gaps may result in replication fork collapse, chromosomal anomalies, and cell death, which contribute to cancer progression and resistance to therapy. Elucidating the processes that avert ssDNA gaps and safeguard replication forks is critical for enhancing cancer treatment approaches by exploiting the vulnerabilities of cancer cells in these pathways

The translesion polymerase Pol Y1 is a constitutive component of the B. subtilis replication machinery.

Unrepaired DNA damage encountered by the cellular replication machinery can stall DNA replication, ultimately leading to cell death. In the DNA damage tolerance pathway translesion synthesis (TLS), replication stalling is alleviated by the recruitment of specialized polymerases to synthesize short stretches of DNA near a lesion. Although TLS promotes cell survival, most TLS polymerases are low-fidelity and must be tightly regulated to avoid harmful mutagenesis. The gram-negative bacterium Escherichia coli has served as the model organism for studies of the molecular mechanisms of bacterial TLS. However, it is poorly understood whether these same mechanisms apply to other bacteria. Here, we use in vivo single-molecule fluorescence microscopy to investigate the TLS polymerase Pol Y1 in the model gram-positive bacterium Bacillus subtilis. We find significant differences in the localization and dynamics of Pol Y1 in comparison to its E. coli homolog, Pol IV. Notably, Pol Y1 is constitutively enriched at or near sites of replication in the absence of DNA damage through interactions with the DnaN clamp; in contrast, Pol IV has been shown to be selectively enriched only upon replication stalling. These results suggest key differences in the roles and mechanisms of regulation of TLS polymerases across different bacterial species.

PARP10 promotes the repair of nascent strand DNA gaps through RAD18 mediated translesion synthesis

Replication stress compromises genomic integrity. Fork blocking lesions such as those induced by cisplatin and other chemotherapeutic agents arrest replication forks. Repriming downstream of these lesions represents an important mechanism of replication restart, however the single stranded DNA (ssDNA) gaps left behind, unless efficiently filled, can serve as entry point for nucleases. Nascent strand gaps can be repaired by BRCA-mediated homology repair. Alternatively, gaps can also be filled by translesion synthesis (TLS) polymerases. How these events are regulated is still not clear. Here, we show that PARP10, a poorly-characterized mono-ADP-ribosyltransferase, is recruited to nascent strand gaps to promote their repair. PARP10 interacts with the ubiquitin ligase RAD18 and recruits it to these structures, resulting in the ubiquitination of the replication factor PCNA. PCNA ubiquitination, in turn, recruits the TLS polymerase REV1 for gap filling. We show that PARP10 recruitment to gaps and the subsequent REV1-mediated gap filling requires both the catalytic activity of PARP10, and its ability to interact with PCNA. We moreover show that PARP10 is hyperactive in BRCA-deficient cells, and its inactivation potentiates gap accumulations and cytotoxicity in these cells. Our work uncovers PARP10 as a regulator of ssDNA gap filling, which promotes genomic stability in BRCA-deficient cells.

Molecular mechanisms of DNA lesion and repair during antibody somatic hypermutation.

Antibody diversification is essential for an effective immune response, with somatic hypermutation (SHM) serving as a key molecular process in this adaptation. Activation-induced cytidine deaminase (AID) initiates SHM by inducing DNA lesions, which are ultimately resolved into point mutations, as well as small insertions and deletions (indels). These mutational outcomes contribute to antibody affinity maturation. The mechanisms responsible for generating point mutations and indels involve the base excision repair (BER) and mismatch repair (MMR) pathways, which are well coordinated to maintain genomic integrity while allowing for beneficial mutations to occur. In this regard, translesion synthesis (TLS) polymerases contribute to the diversity of mutational outcomes in antibody genes by enabling the bypass of DNA lesions. This review summarizes our current understanding of the distinct molecular mechanisms that generate point mutations and indels during SHM. Understanding these mechanisms is critical for elucidating the development of broadly neutralizing antibodies (bnAbs) and autoantibodies, and has implications for vaccine design and therapeutics.

Human translesion DNA polymerases ι and κ mediate tolerance to temozolomide in MGMT-deficient glioblastoma cells.

Glioblastoma (GBM) is a highly aggressive brain tumor associated with poor patient survival. The current standard treatment involves invasive surgery, radiotherapy, and chemotherapy employing temozolomide (TMZ). Resistance to TMZ is, however, a major challenge. Previous work from our group has identified candidate genes linked to TMZ resistance, including genes encoding translesion synthesis (TLS) DNA polymerases iota (Polɩ) and kappa (Polκ). These specialized enzymes are known for bypassing lesions and tolerating DNA damage. Here, we investigated the roles of Polɩ and Polκ in TMZ resistance, employing MGMT-deficient U251-MG glioblastoma cells, with knockout of either POLI or POLK genes encoding Polɩ and Polκ, respectively, and assess their viability and genotoxic stress responses upon subsequent TMZ treatment. Cells lacking either of these polymerases exhibited a significant decrease in viability following TMZ treatment compared to parental counterparts. The restoration of the missing polymerase led to a recovery of cell viability. Furthermore, knockout cells displayed increased cell cycle arrest, mainly in late S-phase, and lower levels of genotoxic stress after TMZ treatment, as assessed by a reduction of γH2AX foci and flow cytometry data. This implies that TMZ treatment does not trigger a significant H2AX phosphorylation response in the absence of these proteins. Interestingly, combining TMZ with Mirin (double-strand break repair pathway inhibitor) further reduced the cell viability and increased DNA damage and γH2AX positive cells in TLS KO cells, but not in parental cells. These findings underscore the crucial roles of Polɩ and Polκ in conferring TMZ resistance and the potential backup role of homologous recombination in the absence of these TLS polymerases. Targeting these TLS enzymes, along with double-strand break DNA repair inhibition, could, therefore, provide a promising strategy to enhance TMZ's effectiveness in treating GBM.

REV1 coordinates a multi-faceted tolerance response to DNA alkylation damage and prevents chromosome shattering in Drosophila melanogaster.

When replication forks encounter damaged DNA, cells utilize damage tolerance mechanisms to allow replication to proceed. These include translesion synthesis at the fork, postreplication gap filling, and template switching via fork reversal or homologous recombination. The extent to which these different damage tolerance mechanisms are utilized depends on cell, tissue, and developmental context-specific cues, the last two of which are poorly understood. To address this gap, we have investigated damage tolerance responses in Drosophila melanogaster. We report that tolerance of DNA alkylation damage in rapidly dividing larval tissues depends heavily on translesion synthesis. Furthermore, we show that the REV1 protein plays a multi-faceted role in damage tolerance in Drosophila. Larvae lacking REV1 are hypersensitive to methyl methanesulfonate (MMS) and have highly elevated levels of γ-H2Av (Drosophila γ-H2AX) foci and chromosome aberrations in MMS-treated tissues. Loss of the REV1 C-terminal domain (CTD), which recruits multiple translesion polymerases to damage sites, sensitizes flies to MMS. In the absence of the REV1 CTD, DNA polymerases eta and zeta become critical for MMS tolerance. In addition, flies lacking REV3, the catalytic subunit of polymerase zeta, require the deoxycytidyl transferase activity of REV1 to tolerate MMS. Together, our results demonstrate that Drosophila prioritize the use of multiple translesion polymerases to tolerate alkylation damage and highlight the critical role of REV1 in the coordination of this response to prevent genome instability.

Protein Assemblies in Translesion Synthesis.

Translesion synthesis (TLS) is a mechanism of DNA damage tolerance utilized by eukaryotic cells to replicate DNA across lesions that impede the high-fidelity replication machinery. In TLS, a series of specialized DNA polymerases are employed, which recognize specific DNA lesions, insert nucleotides across the damage, and extend the distorted primer-template. This allows cells to preserve genetic integrity at the cost of mutations. In humans, TLS enzymes include the Y-family, inserter polymerases, Polη, Polι, Polκ, Rev1, and the B-family extender polymerase Polζ, while in S. cerevisiae only Polη, Rev1, and Polζ are present. To bypass DNA lesions, TLS polymerases cooperate, assembling into a complex on the eukaryotic sliding clamp, PCNA, termed the TLS mutasome. The mutasome assembly is contingent on protein-protein interactions (PPIs) between the modular domains and subunits of TLS enzymes, and their interactions with PCNA and DNA. While the structural mechanisms of DNA lesion bypass by the TLS polymerases and PPIs of their individual modules are well understood, the mechanisms by which they cooperate in the context of TLS complexes have remained elusive. This review focuses on structural studies of TLS polymerases and describes the case of TLS holoenzyme assemblies in action emerging from recent high-resolution Cryo-EM studies.

Methylation and hydroxymethylation of cytosine alter activity and fidelity of translesion DNA polymerases

Epigenetic cytosine methylation covers most of genomic CpG dinucleotides in human cells. In addition to common deamination-mediated mutagenesis at CpG sites, an alternative deamination-independent pathway associated with DNA polymerase activity was previously described. This mutagenesis is characterized by the TCG→TTG mutational signature and is believed to arise from dAMP misincorporation opposite 5-methylcytosine (mC) or its oxidized derivative 5-hydroxymethylcytosine (hmC) by B-family replicative DNA polymerases with disrupted proofreading 3→5′-exonuclease activity. In addition to being less stable and pro-mutagenic themselves, cytosine modifications also increase the risk of adjacent nucleotides damage, including the formation of 8-oxo-2'-deoxyguanosine (8-oxoG), a well-known mutagenic lesion. The effect of cytosine methylation on error-prone DNA polymerases lacking proofreading activity and involved in repair and DNA translesion synthesis remains unexplored. Here we analyze the efficiency and fidelity of translesion Y-family polymerases (Pol κ, Pol η, Pol ι and REV1) and primase-polymerase PrimPol opposite mC and hmC as well as opposite 8-oxoG adjacent to mC in the TCG context. We demonstrate that epigenetic cytosine modifications suppress Pol ι and REV1 activities and lead to increasing dAMP misincorporation by PrimPol, Pol κ and Pol ι in vitro. Cytosine methylation also increases misincorporation of dAMP opposite the adjacent 8-oxoG by PrimPol, decreases the TLS activity of Pol η opposite the lesion but increases dCMP incorporation opposite 8-oxoG by REV1. Altogether, these data suggest that methylation and hydroxymethylation of cytosine alter activity and fidelity of translesion DNA polymerases.

Dual role of proliferating cell nuclear antigen monoubiquitination in facilitating Fanconi anemia-mediated interstrand crosslink repair.

The Fanconi anemia (FA) repair pathway governs repair of highly genotoxic DNA interstrand crosslinks (ICLs) and relies on translesion synthesis (TLS). TLS is facilitated by REV1 or site-specific monoubiquitination of proliferating cell nuclear antigen (PCNA) (PCNA-Ub) at lysine 164 (K164). A PcnaK164R/K164R but not Rev1-/- mutation renders mammals hypersensitive to ICLs. Besides the FA pathway, alternative pathways have been associated with ICL repair (1, 2), though the decision making between those remains elusive. To study the dependence and relevance of PCNA-Ub in FA repair, we intercrossed PcnaK164R/+; Fancg-/+ mice. A combined mutation (PcnaK164R/K164R; Fancg-/- ) was found embryonically lethal. RNA-seq of primary double-mutant (DM) mouse embryonic fibroblasts (MEFs) revealed elevated levels of replication stress-induced checkpoints. To exclude stress-induced confounders, we utilized a Trp53 knock-down to obtain a model to study ICL repair in depth. Regarding ICL-induced cell toxicity, cell cycle arrest, and replication fork progression, single-mutant and DM MEFs were found equally sensitive, establishing PCNA-Ub to be critical for FA-ICL repair. Immunoprecipitation and spectrometry-based analysis revealed an unknown role of PCNA-Ub in excluding mismatch recognition complex MSH2/MSH6 from being recruited to ICLs. In conclusion, our results uncovered a dual function of PCNA-Ub in ICL repair, i.e. exclude MSH2/MSH6 recruitment to channel the ICL toward canonical FA repair, in addition to its established role in coordinating TLS opposite the unhooked ICL.