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Azide-alkyne cycloaddition ("click chemistry") has found wide use in the analysis of molecular interactions in living cells. 5-ethynyl-2-(hydroxymethyl)tetrahydrofuran-3-ol (EAP) is a recently developed apurinic/apyrimidinic (AP) site analog functionalized with an ethynyl moiety, which can be introduced into cells in DNA constructs to perform labeling or cross-linking in situ. However, as a non-natural nucleoside, EAP could be subject to removal by DNA repair and misreading by DNA polymerases. Here, we investigate the interaction of this clickable AP site analog with DNA polymerases and base excision repair enzymes. Similarly to the natural AP site, EAP was non-instructive and followed the "A-rule", directing residual but easily detectable incorporation of dAMP by E. coli DNA polymerase I Klenow fragment, bacteriophage RB69 DNA polymerase and human DNA polymerase β. On the contrary, EAP was blocking for DNA polymerases κ and λ. EAP was an excellent substrate for the major human AP endonuclease APEX1 and E. coli AP exonucleases Xth and Nfo but was resistant to the AP lyase activity of DNA glycosylases. Overall, our data indicate that EAP, once within a cell, would represent a replication block and would be removed through an AP endonuclease-initiated long-patch base excision repair pathway.

G-quadruplexes (G4s), a type of non-B DNA, play important roles in a wide range of molecular processes, including replication, transcription, and translation. Genome integrity relies on efficient and accurate DNA synthesis, and is compromised by various stressors, to which non-B DNA structures such as G4s can be particularly vulnerable. However, the impact of G4 structures on DNA polymerase fidelity is largely unknown. Using an in vitro forward mutation assay, we investigated the fidelity of human DNA polymerases delta (δ4, four-subunit), eta (η), and kappa (κ) during synthesis of G4 motifs representing those in the human genome. The motifs differ in sequence, topology, and stability, features that may affect DNA polymerase errors. Polymerase error rate hierarchy (δ4 < κ < η) is largely maintained during G4 synthesis. Importantly, we observed unique polymerase error signatures during synthesis of VEGF G4 motifs, stable G4s which form parallel topologies. These statistically significant errors occurred within, immediately flanking, and encompassing the G4 motif. For pol δ4, the errors were deletions, insertions and complex errors within the G4 or encompassing the G4 motif and surrounding sequence. For pol η, the errors occurred in 3' sequences flanking the G4 motif. For pol κ, the errors were frameshift mutations within G-tracts of the G4. Because these error signatures were not observed during synthesis of an antiparallel G4 and, to a lesser extent, a hybrid G4, we suggest that G4 topology and/or stability could influence polymerase fidelity. Using in silico analyses, we show that most polymerase errors are predicted to have minimal effects on predicted G4 stability. Our results provide a unique view of G4s not previously elucidated, showing that G4 motif heterogeneity differentially influences polymerase fidelity within the motif and flanking sequences. Thus, our study advances the understanding of how DNA polymerase errors contribute to G4 mutagenesis., Competing Interests: Conflict of interest statement The authors declare no conflict of interest with this research., (Copyright © 2022 The Authors. Published by Elsevier B.V. All rights reserved.)

Methods for the real-time monitoring of the substrate acceptance of modified nucleotides by DNA polymerases are in high demand. In a step towards this aim, we have incorporated ferrocene-based abasic nucleotides into DNA templates and evaluated their compatibility with enzymatic synthesis of unmodified and modified DNA. All canonical nucleotides can be incorporated opposite ferrocene sites with a strong preference for purines. DNA polymerases with lesion-bypass capacity such as Dpo4 allow DNA synthesis to be resumed beyond the site of incorporation. Modified purine nucleotides can readily be incorporated opposite ferrocene basic site analogs, while pyrimidine nucleotides decorated with simple side-chains are also readily tolerated. These findings open up directions for the design of electrochemical sensing devices for the monitoring of enzymatic synthesis of natural or modified DNA.

Five 2'-deoxyribonucleoside triphosphates (dNTPs) derived from epigenetic pyrimidines (5-methylcytosine, 5-hydroxymethylcytosine, 5-formylcytosine, 5-hydroxymethyluracil, and 5-formyluracil) were prepared and systematically studied as substrates for nine DNA polymerases in competition with natural dNTPs by primer extension experiments. The incorporation of these substrates was evaluated by a restriction endonucleases cleavage-based assay and by a kinetic study of single nucleotide extension. All of the modified pyrimidine dNTPs were good substrates for the studied DNA polymerases that incorporated a significant percentage of the modified nucleotides into DNA even in the presence of natural nucleotides. 5-Methylcytosine dNTP was an even better substrate for most polymerases than natural dCTP. On the other hand, 5-hydroxymethyl-2'-deoxyuridine triphosphate was not the best substrate for SPO1 DNA polymerase, which naturally synthesizes 5hmU-rich genomes of the SPO1 bacteriophage. The results shed light onto the possibility of gene silencing through recycling and random incorporation of epigenetic nucleotides and into the replication of modified bacteriophage genomes.

DNA repair pathway inhibitors are a new class of anticancer drugs that are advancing in clinical trials. Peposertib is an inhibitor of DNA-dependent protein kinase (DNA-PK), which is a key driver of nonhomologous end-joining (NHEJ). To identify regulators of response to peposertib, we performed a genome-wide CRISPR knockout screen and found that loss of POLQ (polymerase theta, POLθ) and other genes in the microhomology-mediated end-joining (MMEJ) pathway are key predictors of sensitivity to DNA-PK inhibition. Simultaneous disruption of two DNA repair pathways via combined treatment with peposertib plus a POLθ inhibitor novobiocin exhibited synergistic synthetic lethality resulting from accumulation of toxic levels of DNA double-strand break end resection. TP53-mutant tumor cells were resistant to peposertib but maintained elevated expression of POLQ and increased sensitivity to novobiocin. Consequently, the combination of peposertib plus novobiocin resulted in synthetic lethality in TP53-deficient tumor cell lines, organoid cultures, and patient-derived xenograft models. Thus, the combination of a targeted DNA-PK/NHEJ inhibitor with a targeted POLθ/MMEJ inhibitor may provide a rational treatment strategy for TP53-mutant solid tumors., Significance: Combined inhibition of NHEJ and MMEJ using two nontoxic, targeted DNA repair inhibitors can effectively induce toxic DNA damage to treat TP53-deficient cancers., (©2022 American Association for Cancer Research.)

Understanding the occurrence, repair, and biological consequences of DNA damage is important in environmental toxicology and risk assessment. The most common way to assess DNA damage elicited by exogenous sources in a laboratory setting is to expose cells or experimental animals with chemicals that modify DNA. Owing to the lack of reaction specificities of DNA damaging agents, the approach frequently does not allow for induction of a specific DNA lesion. Herein, we employed metabolic labeling to selectively incorporate N 2 -methyl-dG ( N 2 -MedG) and N 2 - n -butyl-dG ( N 2 - n BudG) into genomic DNA of cultured mammalian cells, and investigated how the levels of the two lesions in cellular DNA are modulated by different DNA repair factors. Our results revealed that nucleotide excision repair (NER) exert moderate effects on the removal of N 2 -MedG and N 2 - n BudG from genomic DNA. We also observed that DNA polymerases κ and η contribute to the incorporation of N 2 -MedG into genomic DNA and modulate its repair in human cells. In addition, loss of ALKBH3 resulted in higher frequencies of N 2 -MedG and N 2 - n BuG incorporation into genomic DNA, suggesting a role of oxidative dealkylation in the reversal of these lesions. Together, our study provided new insights into the repair of minor-groove N 2 -alkyl-dG lesions in mammalian cells.

DNA polymerase theta (Polθ) is an attractive synthetic lethal target for drug discovery, predicted to be efficacious against breast and ovarian cancers harboring BRCA-mutant alleles. Here, we describe our hit-to-lead efforts in search of a selective inhibitor of human Polθ (encoded by POLQ). A high-throughput screening campaign of 350,000 compounds identified an 11 micromolar hit, giving rise to the N2-substituted fused pyrazolo series, which was validated by biophysical methods. Structure-based drug design efforts along with optimization of cellular potency and ADME ultimately led to the identification of RP-6685 : a potent, selective, and orally bioavailable Polθ inhibitor that showed in vivo efficacy in an HCT116 BRCA2 -/- mouse tumor xenograft model.

Mismatch repair (MMR) is a replication-coupled DNA repair mechanism and plays multiple roles at the replication fork. The well-established MMR functions include correcting misincorporated nucleotides that have escaped the proofreading activity of DNA polymerases, recognizing nonmismatched DNA adducts, and triggering a DNA damage response. In an attempt to determine whether MMR regulates replication progression in cells expressing an ultramutable DNA polymerase ɛ (Polɛ), carrying a proline-to-arginine substitution at amino acid 286 (Polɛ-P286R), we identified an unusual MMR function in response to hydroxyurea (HU)-induced replication stress. Polɛ-P286R cells treated with hydroxyurea exhibit increased MRE11-catalyzed nascent strand degradation. This degradation by MRE11 depends on the mismatch recognition protein MutSα and its binding to stalled replication forks. Increased MutSα binding at replication forks is also associated with decreased loading of replication fork protection factors FANCD2 and BRCA1, suggesting blockage of these fork protection factors from loading to replication forks by MutSα. We find that the MutSα-dependent MRE11-catalyzed fork degradation induces DNA breaks and various chromosome abnormalities. Therefore, unlike the well-known MMR functions of ensuring replication fidelity, the newly identified MMR activity of promoting genome instability may also play a role in cancer avoidance by eliminating rogue cells.

In bacterial cells, DNA damage tolerance is manifested by the action of translesion DNA polymerases that can synthesize DNA across template lesions that typically block the replicative DNA polymerase III. It has been suggested that one of these translesion DNA synthesis DNA polymerases, DNA polymerase IV, can either act in concert with the replisome, switching places on the β sliding clamp with DNA polymerase III to bypass the template damage, or act subsequent to the replisome skipping over the template lesion in the gap in nascent DNA left behind as the replisome continues downstream. Evidence exists in support of both mechanisms. Using single-molecule analyses, we show that DNA polymerase IV associates with the replisome in a concentration-dependent manner and remains associated over long stretches of replication fork progression under unstressed conditions. This association slows the replisome, requires DNA polymerase IV binding to the β clamp but not its catalytic activity, and is reinforced by the presence of the γ subunit of the β clamp-loading DnaX complex in the DNA polymerase III holoenzyme. Thus, DNA damage is not required for association of DNA polymerase IV with the replisome. We suggest that under stress conditions such as induction of the SOS response, the association of DNA polymerase IV with the replisome provides both a surveillance/bypass mechanism and a means to slow replication fork progression, thereby reducing the frequency of collisions with template damage and the overall mutagenic potential.

DNA lesions in S phase threaten genome stability. The DNA damage tolerance (DDT) pathways overcome these obstacles and allow completion of DNA synthesis by the use of specialised translesion (TLS) DNA polymerases or through recombination-related processes. However, how these mechanisms coordinate with each other and with bulk replication remains elusive. To address these issues, we monitored the variation of replication intermediate architecture in response to ultraviolet irradiation using transmission electron microscopy. We show that the TLS polymerase η, able to accurately bypass the major UV lesion and mutated in the skin cancer-prone xeroderma pigmentosum variant (XPV) syndrome, acts at the replication fork to resolve uncoupling and prevent post-replicative gap accumulation. Repriming occurs as a compensatory mechanism when this on-the-fly mechanism cannot operate, and is therefore predominant in XPV cells. Interestingly, our data support a recombination-independent function of RAD51 at the replication fork to sustain repriming. Finally, we provide evidence for the post-replicative commitment of recombination in gap repair and for pioneering observations of in vivo recombination intermediates. Altogether, we propose a chronology of UV damage tolerance in human cells that highlights the key role of polη in shaping this response and ensuring the continuity of DNA synthesis., (© The Author(s) 2022. Published by Oxford University Press on behalf of Nucleic Acids Research.)

ATR kinase is a central regulator of the DNA damage response (DDR) and cell cycle checkpoints. ATR kinase inhibitors (ATRi's) combine with radiation to generate CD8 + T cell-dependent responses in mouse models of cancer. We show that ATRi's induce cyclin-dependent kinase 1 (CDK1)-dependent origin firing across active replicons in CD8 + T cells activated ex vivo while simultaneously decreasing the activity of rate-limiting enzymes for nucleotide biosynthesis. These pleiotropic effects of ATRi induce deoxyuridine (dU) contamination in genomic DNA, R loops, RNA-DNA polymerase collisions, and interferon-α/β (IFN-α/β). Remarkably, thymidine rescues ATRi-induced dU contamination and partially rescues death and IFN-α/β expression in proliferating CD8 + T cells. Thymidine also partially rescues ATRi-induced cancer cell death. We propose that ATRi-induced dU contamination contributes to dose-limiting leukocytopenia and inflammation in the clinic and CD8 + T cell-dependent anti-tumor responses in mouse models. We conclude that ATR is essential to limit dU contamination in genomic DNA and IFN-α/β expression., Competing Interests: Declarations of interests The authors declare no competing interests., (Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.)

Recent advances in next generation sequencing technologies enable reading DNA molecules hundreds of kilobases in length and motivate development of DNA amplification methods capable of producing long amplicons. In vivo, DNA replication is performed not by a single polymerase enzyme, but multiprotein complexes called replisomes. Here, we investigate strand-displacement amplification reactions using the T7 replisome, a macromolecular complex of a helicase, a single-stranded DNA binding protein, and a DNA polymerase. The T7 replisome may initiate processive DNA synthesis from DNA nicks, and the reaction of a 48 kilobase linear double stranded DNA substrate with the T7 replisome and nicking endonucleases is shown to produce discrete DNA amplicons. To gain a mechanistic understanding of this reaction, we utilized Oxford Nanopore long-read sequencing technology. Sequence analysis of the amplicons revealed chimeric DNA reads and uncovered a connection between template switching and polymerase exonuclease activity. Nanopore sequencing provides insight to guide the further development of isothermal amplification methods for long DNA, and our results highlight the need for high-specificity, high-turnover nicking endonucleases to initiate DNA amplification without thermal denaturation., Competing Interests: The authors are employees of New England Biolabs, manufacturer of some enzymes used in the manuscript. A provisional patent application has been filed related to some of the results described in this work. This does not alter our adherence to PLOS ONE policies on sharing data and materials.

Protection of stalled replication forks is essential to prevent genome instability, a major driving force of tumorigenesis. Several key regulators of DNA double-stranded break (DSB) repair, including 53BP1 and RIF1, have been implicated in fork protection. MAD2L2, also known as REV7, plays an important role downstream of 53BP1/RIF1 by counteracting resection at DSBs in the recently discovered shieldin complex. The ability to bind and counteract resection at exposed DNA ends at DSBs makes MAD2L2/shieldin a prime candidate for also suppressing nucleolytic processing at stalled replication forks. However, the function of MAD2L2/shieldin outside of DNA repair is unknown. Here we address this by using genetic and single-molecule analyses and find that MAD2L2 is required for protecting and restarting stalled replication forks. MAD2L2 loss leads to uncontrolled MRE11-dependent resection of stalled forks and single-stranded DNA accumulation, which causes irreparable genomic damage. Unexpectedly, MAD2L2 limits resection at stalled forks independently of shieldin, since fork protection remained unaffected by shieldin loss. Instead, MAD2L2 cooperates with the DNA polymerases REV3L and REV1 to promote fork stability. Thus, MAD2L2 suppresses aberrant nucleolytic processing both at DSBs and stalled replication forks by differentially engaging shieldin and REV1/REV3L, respectively., (© 2022. The Author(s).)

DNA polymerase ζ (pol ζ) plays a central role in replicating damaged genomic DNA. When DNA synthesis stalls at a lesion, it participates in translesion DNA synthesis (TLS), which helps replication proceed. TLS prevents cell death at the expense of new mutations. The current model indicates that pol ζ-dependent TLS events are mediated by Pol31/Pol32 pol ζ subunits, which are shared with replicative polymerase pol δ. Surprisingly, we found that the mutant rev3- Δ C in yeast, which lacks the C-terminal domain (CTD) of the catalytic subunit of pol ζ and, thus, the platform for interaction with Pol31/Pol32, retains most pol ζ functions. To understand the underlying mechanisms, we studied TLS in normal templates or templates with abasic sites in vitro in primer extension reactions with purified four-subunit pol ζ versus pol ζ with Rev3-ΔC. We also examined the specificity of ultraviolet radiation (UVR)-induced mutagenesis in the rev3- Δ C strains. We found that the absence of Rev3 CTD reduces activity levels, but does not alter the basic biochemical properties of pol ζ, and alters the mutation spectrum only at high doses of UVR, alluding to the existence of mechanisms of recruitment of pol ζ to UVR-damaged sites independent of the interaction of Pol31/Pol32 with the CTD of Rev3.

Pseudorabies virus (PRV) is a ubiquitous and economically important swine alphaherpesvirus that causes devastating swine diseases worldwide. PRV-encoded DNA-dependent DNA polymerase, comprised of the catalytic subunit UL30 and the accessory subunit UL42, is essential for viral replication. PRV UL30 and UL42 act as a heterodimer with UL30 harboring inherent DNA polymerase activity and UL42 conferring processivity on the DNA polymerase holoenzyme. The formation of PRV UL30/UL42 heterodimer holoenzyme through protein-protein interactions is indispensable for viral replication. In work described here, we defined the key domains that mediate PRV UL30/UL42 interaction, and found that the 41 carboxy-terminal amino acids region of PRV UL30 is critical for its interaction with UL42. Intriguingly, a synthetic peptide corresponding to these 41 carboxy-terminal amino acid residues efficiently disrupted PRV UL30/UL42 interaction through competitively binding to UL42. These findings suggest that the peptides from the PRV DNA polymerase UL30/UL42 subunit interface may represent potential targets for designing a novel intervention strategy against PRV infection. This work further strengthens the concept that the herpesvirus DNA polymerase catalytic subunits utilize their extreme carboxy-terminal domains as a conserved mechanism to associate with their cognate accessory subunits, providing us the opportunity of designing novel antiviral agents against herpesvirus infection through disruption of the herpesvirus DNA polymerase subunit interactions., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Processivity clamps tether DNA polymerases to DNA, allowing their access to the primer-template junction. In addition to DNA replication, DNA polymerases also participate in various genome maintenance activities, including translesion synthesis (TLS). However, owing to the error-prone nature of TLS polymerases, their association with clamps must be tightly regulated. Here we show that fork-associated ssDNA-binding protein (SSB) selectively enriches the bacterial TLS polymerase Pol IV at stalled replication forks. This enrichment enables Pol IV to associate with the processivity clamp and is required for TLS on both the leading and lagging strands. In contrast, clamp-interacting proteins (CLIPs) lacking SSB binding are spatially segregated from the replication fork, minimally interfering with Pol IV-mediated TLS. We propose that stalling-dependent structural changes within clusters of fork-associated SSB establish hierarchical access to the processivity clamp. This mechanism prioritizes a subset of CLIPs with SSB-binding activity and facilitates their exchange at the replication fork., (© 2022. The Author(s), under exclusive licence to Springer Nature America, Inc.)

DNA polymerase (Pol) ν and Pol θ are two specialized A-family DNA polymerases that function in the translesion synthesis of certain DNA lesions. However, the biological functions of human Pols ν and θ in cellular replicative bypass of 8-oxo-7,8-dihydroguanine (8-oxoG), an important carcinogenesis-related biomarker of oxidative DNA damage, remain unclear. Herein, we showed that depletion of Pols ν and θ in human cells could cause an elevated hypersensitivity to oxidative stress induced by hydrogen peroxide. Using next-generation sequencing-based lesion bypass and mutagenesis assay, we further demonstrated that Pols ν and θ had important roles in promoting translesion synthesis of 8-oxoG in human cells. We also found that the depletion of Pol ν, but not Pol θ, caused a substantial reduction in G → T mutation frequency for 8-oxoG. These findings provided novel insights into the involvement of A-family DNA polymerases in oxidative DNA damage response.

Human cells encode up to 15 DNA polymerases with specialized functions in chromosomal DNA synthesis and damage repair. In contrast, complex DNA viruses, such as those of the herpesviridae family, encode a single B-family DNA polymerase. This disparity raises the possibility that DNA viruses may rely on host polymerases for synthesis through complex DNA geometries. We tested the importance of error-prone Y-family polymerases involved in translesion synthesis (TLS) to human cytomegalovirus (HCMV) infection. We find most Y-family polymerases involved in the nucleotide insertion and bypass of lesions restrict HCMV genome synthesis and replication. In contrast, other TLS polymerases, such as the polymerase ζ complex, which extends past lesions, was required for optimal genome synthesis and replication. Depletion of either the polζ complex or the suite of insertion polymerases demonstrate that TLS polymerases suppress the frequency of viral genome rearrangements, particularly at GC-rich sites and repeat sequences. Moreover, while distinct from HCMV, replication of the related herpes simplex virus type 1 is impacted by host TLS polymerases, suggesting a broader requirement for host polymerases for DNA virus replication. These findings reveal an unexpected role for host DNA polymerases in ensuring viral genome stability.

Translesion synthesis (TLS) is a cell signaling pathway that facilitates the tolerance of replication stress. Increased TLS activity, the particularly elevated expression of TLS polymerases, has been linked to resistance to cancer chemotherapeutics and significantly altered patient outcomes. Building upon current knowledge, we found that the expression of one of these TLS polymerases ( POLI ) is associated with significant differences in cervical and pancreatic cancer survival. These data led us to hypothesize that POLI expression is associated with cancer survival more broadly. However, when cancers were grouped cancer type, POLI expression did not have a significant prognostic value. We presented a binary cancer random forest classifier using 396 genes that influence the prognostic characteristics of POLI in cervical and pancreatic cancer selected via graphical least absolute shrinkage and selection operator. The classifier was then used to cluster patients with bladder, breast, colorectal, head and neck, liver, lung, ovary, melanoma, stomach, and uterus cancer when high POLI expression was associated with worsened survival (Group I) or with improved survival (Group II). This approach allowed us to identify cancers where POLI expression is a significant prognostic factor for survival ( p = 0.028 in Group I and p = 0.0059 in Group II). Multiple independent validation approaches, including the gene ontology enrichment analysis and visualization tool and network visualization support the classification scheme. The functions of the selected genes involving mitochondrial translational elongation, Wnt signaling pathway, and tumor necrosis factor-mediated signaling pathway support their association with TLS and replication stress. Our multidisciplinary approach provides a novel way of identifying tumors where increased TLS polymerase expression is associated with significant differences in cancer survival.

In human cells, ATP is generated using oxidative phosphorylation machinery, which is inoperable without proteins encoded by mitochondrial DNA (mtDNA). The DNA polymerase gamma (Polγ) repairs and replicates the multicopy mtDNA genome in concert with additional factors. The Polγ catalytic subunit is encoded by the POLG gene, and mutations in this gene cause mtDNA genome instability and disease. Barriers to studying the molecular effects of disease mutations include scarcity of patient samples and a lack of available mutant models; therefore, we developed a human SJCRH30 myoblast cell line model with the most common autosomal dominant POLG mutation, c.2864A>G/p.Y955C, as individuals with this mutation can present with progressive skeletal muscle weakness. Using on-target sequencing, we detected a 50% conversion frequency of the mutation, confirming heterozygous Y955C substitution. We found mutated cells grew slowly in a glucose-containing medium and had reduced mitochondrial bioenergetics compared with the parental cell line. Furthermore, growing Y955C cells in a galactose-containing medium to obligate mitochondrial function enhanced these bioenergetic deficits. Also, we show complex I NDUFB8 and ND3 protein levels were decreased in the mutant cell line, and the maintenance of mtDNA was severely impaired (i.e., lower copy number, fewer nucleoids, and an accumulation of Y955C-specific replication intermediates). Finally, we show the mutant cells have increased sensitivity to the mitochondrial toxicant 2'-3'-dideoxycytidine. We expect this POLG Y955C cell line to be a robust system to identify new mitochondrial toxicants and therapeutics to treat mitochondrial dysfunction., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

DNA damage tolerance pathways are regulated by proliferating cell nuclear antigen (PCNA) modifications at lysine 164. Translesion DNA synthesis by DNA polymerase η (Polη) is well studied, but less is known about Polη-independent mechanisms. Illudin S and its derivatives induce alkyl DNA adducts, which are repaired by transcription-coupled nucleotide excision repair (TC-NER). We demonstrate that in addition to TC-NER, PCNA modification at K164 plays an essential role in cellular resistance to these compounds by overcoming replication blockages, with no requirement for Polη. Polκ and RING finger and WD repeat domain 3 (RFWD3) contribute to tolerance, and are both dependent on PCNA modifications. Although RFWD3 is a FANC protein, we demonstrate that it plays a role in DNA damage tolerance independent of the FANC pathway. Finally, we demonstrate that RFWD3-mediated cellular survival after UV irradiation is dependent on PCNA modifications but is independent of Polη. Thus, RFWD3 contributes to PCNA modification-dependent DNA damage tolerance in addition to translesion DNA polymerases., (© 2022 Kanao et al.)

Crosslink repair depends on the Fanconi anemia pathway and translesion synthesis polymerases that replicate over unhooked crosslinks. Translesion synthesis is regulated via ubiquitination of PCNA, and independently via translesion synthesis polymerase REV1. The division of labor between PCNA-ubiquitination and REV1 in interstrand crosslink repair is unclear. Inhibition of either of these pathways has been proposed as a strategy to increase cytotoxicity of platinating agents in cancer treatment. Here, we defined the importance of PCNA-ubiquitination and REV1 for DNA in mammalian ICL repair. In mice, loss of PCNA-ubiquitination, but not REV1, resulted in germ cell defects and hypersensitivity to cisplatin. Loss of PCNA-ubiquitination, but not REV1 sensitized mammalian cancer cell lines to cisplatin. We identify polymerase Kappa as essential in tolerating DNA damage-induced lesions, in particular cisplatin lesions. Polk-deficient tumors were controlled by cisplatin treatment and it significantly delayed tumor outgrowth and increased overall survival of tumor bearing mice. Our results indicate that PCNA-ubiquitination and REV1 play distinct roles in DNA damage tolerance. Moreover, our results highlight POLK as a critical TLS polymerase in tolerating multiple genotoxic lesions, including cisplatin lesions. The relative frequent loss of Polk in cancers indicates an exploitable vulnerability for precision cancer medicine., (© The Author(s) 2022. Published by Oxford University Press on behalf of Nucleic Acids Research.)

We present a structural and functional analysis of the DNA polymerase of thermophilic Thermus thermophilus MAT72 phage vB&#95;Tt72. The enzyme shows low sequence identity (<30%) to the members of the type-A family of DNA polymerases, except for two yet uncharacterized DNA polymerases of T. thermophilus phages: φYS40 (91%) and φTMA (90%). The Tt72 polA gene does not complement the Escherichia colipolA− mutant in replicating polA-dependent plasmid replicons. It encodes a 703-aa protein with a predicted molecular weight of 80,490 and an isoelectric point of 5.49. The enzyme contains a nucleotidyltransferase domain and a 3′-5′ exonuclease domain that is engaged in proofreading. Recombinant enzyme with His-tag at the N-terminus was overproduced in E. coli, subsequently purified by immobilized metal affinity chromatography, and biochemically characterized. The enzyme exists in solution in monomeric form and shows optimum activity at pH 8.5, 25 mM KCl, and 0.5 mM Mg2+. Site-directed analysis proved that highly-conserved residues D15, E17, D78, D180, and D184 in 3′-5′ exonuclease and D384 and D615 in the nucleotidyltransferase domain are critical for the enzyme’s activity. Despite the source of origin, the Tt72 DNA polymerase has not proven to be highly thermoresistant, with a temperature optimum at 55 °C. Above 60 °C, the rapid loss of function follows with no activity > 75 °C. However, during heat treatment (10 min at 75 °C), trehalose, trimethylamine N-oxide, and betaine protected the enzyme against thermal inactivation. A midpoint of thermal denaturation at Tm = 74.6 °C (ΔHcal = 2.05 × 104 cal mol−1) and circular dichroism spectra > 60 °C indicate the enzyme’s moderate thermal stability.

DNA polymerases have evolved to feature a highly conserved activity across the tree of life: formation of, without exception, internucleotidyl O-P linkages. Can this linkage selectivity be overcome by design to produce xenonucleic acids? Here, we report that the structure-guided redesign of an archaeal DNA polymerase, 9°N, exhibits a new activity undetectable in the wild-type enzyme: catalyzing the formation of internucleotidyl N-P linkages using 3'-NH 2 -ddNTPs. Replacing a metal-binding aspartate in the 9°N active site with asparagine was key to the emergence of this unnatural enzyme activity. MD simulations provided insights into how a single substitution enhances the productive positioning of a 3'-amino nucleophile in the active site. Further remodeling of the protein-nucleic acid interface in the finger subdomain yielded a quadruple-mutant variant (9°N-NRQS) displaying DNA-dependent NP-DNA polymerase activity. In addition, the engineered promiscuity of 9°N-NRQS was leveraged for one-pot synthesis of DNA─NP-DNA copolymers. This work sheds light on the molecular basis of substrate fidelity and latent promiscuity in enzymes.

Despite excellent vaccines, resurgent outbreaks of hepatitis A have caused thousands of hospitalizations and hundreds of deaths within the United States in recent years. There is no effective antiviral therapy for hepatitis A, and many aspects of the hepatitis A virus (HAV) replication cycle remain to be elucidated. Replication requires the zinc finger protein ZCCHC14 and noncanonical TENT4 poly(A) polymerases with which it associates, but the underlying mechanism is unknown. Here, we show that ZCCHC14 and TENT4A/B are required for viral RNA synthesis following translation of the viral genome in infected cells. Cross-linking immunoprecipitation sequencing (CLIP-seq) experiments revealed that ZCCHC14 binds a small stem-loop in the HAV 5' untranslated RNA possessing a Smaug recognition-like pentaloop to which it recruits TENT4. TENT4 polymerases lengthen and stabilize the 3' poly(A) tails of some cellular and viral mRNAs, but the chemical inhibition of TENT4A/B with the dihydroquinolizinone RG7834 had no impact on the length of the HAV 3' poly(A) tail, stability of HAV RNA, or cap-independent translation of the viral genome. By contrast, RG7834 inhibited the incorporation of 5-ethynyl uridine into nascent HAV RNA, indicating that TENT4A/B function in viral RNA synthesis. Consistent with potent in vitro antiviral activity against HAV (IC 50 6.11 nM), orally administered RG7834 completely blocked HAV infection in Ifnar1 -/- mice, and sharply reduced serum alanine aminotransferase activities, hepatocyte apoptosis, and intrahepatic inflammatory cell infiltrates in mice with acute hepatitis A. These results reveal requirements for ZCCHC14-TENT4A/B in hepatovirus RNA synthesis, and suggest that TENT4A/B inhibitors may be useful for preventing or treating hepatitis A in humans.

Homologs of the mutagenic Escherichia coli DNA polymerase V (pol V) are encoded by numerous pathogens and mobile elements. We have used Rum pol (RumA'2B), from the integrative conjugative element (ICE), R391, as a model mobile element-encoded polymerase (MEPol). The highly mutagenic Rum pol is transferred horizontally into a variety of recipient cells, including many pathogens. Moving between species, it is unclear if Rum pol can function on its own or requires activation by host factors. Here, we show that Rum pol biochemical activity requires the formation of a physical mutasomal complex, Rum Mut, containing RumA'2B-RecA-ATP, with RecA being donated by each recipient bacteria. For R391, Rum Mut specific activities in vitro and mutagenesis rates in vivo depend on the phylogenetic distance of host-cell RecA from E. coli RecA. Rum pol is a highly conserved and effective mobile catalyst of rapid evolution, with the potential to generate a broad mutational landscape that could serve to ensure bacterial adaptation in antibiotic-rich environments leading to the establishment of antibiotic resistance., (© The Author(s) 2022. Published by Oxford University Press on behalf of Nucleic Acids Research.)

The APOBEC3 family of cytosine deaminases has been implicated in some of the most prevalent mutational signatures in cancer 1-3 . However, a causal link between endogenous APOBEC3 enzymes and mutational signatures in human cancer genomes has not been established, leaving the mechanisms of APOBEC3 mutagenesis poorly understood. Here, to investigate the mechanisms of APOBEC3 mutagenesis, we deleted implicated genes from human cancer cell lines that naturally generate APOBEC3-associated mutational signatures over time 4 . Analysis of non-clustered and clustered signatures across whole-genome sequences from 251 breast, bladder and lymphoma cancer cell line clones revealed that APOBEC3A deletion diminished APOBEC3-associated mutational signatures. Deletion of both APOBEC3A and APOBEC3B further decreased APOBEC3 mutation burdens, without eliminating them. Deletion of APOBEC3B increased APOBEC3A protein levels, activity and APOBEC3A-mediated mutagenesis in some cell lines. The uracil glycosylase UNG was required for APOBEC3-mediated transversions, whereas the loss of the translesion polymerase REV1 decreased overall mutation burdens. Together, these data represent direct evidence that endogenous APOBEC3 deaminases generate prevalent mutational signatures in human cancer cells. Our results identify APOBEC3A as the main driver of these mutations, indicate that APOBEC3B can restrain APOBEC3A-dependent mutagenesis while contributing its own smaller mutation burdens and dissect mechanisms that translate APOBEC3 activities into distinct mutational signatures., (© 2022. The Author(s).)

The X family polymerases (PolXs) are specialized DNA polymerases that are found in all domains of life. While the main representatives of eukaryotic PolXs, which have dedicated functions in DNA repair, were studied in much detail, the functions and diversity of prokaryotic PolXs have remained largely unexplored. Here, by combining a comprehensive bioinformatic analysis of prokaryotic PolXs and biochemical experiments involving selected recombinant enzymes, we reveal a previously unrecognized group of PolXs that seem to be lacking DNA polymerase activity. The noncanonical PolXs contain substitutions of the key catalytic residues and deletions in their polymerase and dNTP binding sites in the palm and fingers domains, but contain functional nuclease domains, similar to canonical PolXs. We demonstrate that representative noncanonical PolXs from the Deinococcus genus are indeed inactive as DNA polymerases but are highly efficient as 3'-5' exonucleases. We show that both canonical and noncanonical PolXs are often encoded together with the components of the non-homologous end joining pathway and may therefore participate in double-strand break repair, suggesting an evolutionary conservation of this PolX function. This is a remarkable example of polymerases that have lost their main polymerase activity, but retain accessory functions in DNA processing and repair., (© The Author(s) 2022. Published by Oxford University Press on behalf of Nucleic Acids Research.)

DNA mismatch repair removes mis-incorporated bases after DNA replication and reduces the error rate a 100-1000-fold. After recognition of a mismatch, a large section of up to a thousand nucleotides is removed from the daughter strand followed by re-synthesis. How these opposite activities are coordinated is poorly understood. Here we show that the Escherichia coli MutL protein binds to the 3' end of the resected strand and blocks access of Pol I and Pol III. The cryo-EM structure of an 85-kDa MutL-DNA complex, determined to 3.7 Å resolution, reveals a unique DNA binding mode that positions MutL at the 3' end of a primer-template, but not at a 5' resected DNA end or a blunt DNA end. Hence, our work reveals a novel role for MutL in the final stages of mismatch repair by preventing premature DNA synthesis during removal of the mismatched strand., (© The Author(s) 2022. Published by Oxford University Press on behalf of Nucleic Acids Research.)

DNA polymerases can process a wide variety of structurally diverse nucleotide substrates, but the molecular basis by which the analogs are processed is not completely understood. Here, we demonstrate the utility of environment-sensitive heterocycle-modified fluorescent nucleotide substrates in probing the incorporation mechanism of DNA polymerases in real time and at the atomic level. The nucleotide analogs containing a selenophene, benzofuran, or benzothiophene moiety at the C5 position of 2'-deoxyuridine are incorporated into oligonucleotides (ONs) with varying efficiency, which depends on the size of the heterocycle modification and the DNA polymerase sequence family used. KlenTaq (A family DNA polymerase) is sensitive to the size of the modification as it incorporates only one heterobicycle-modified nucleotide into the growing polymer, whereas it efficiently incorporates the selenophene-modified nucleotide analog at multiple positions. Notably, in the single nucleotide incorporation assay, irrespective of the heterocycle size, it exclusively adds a single nucleotide at the 3'-end of a primer, which enabled devising a simple two-step site-specific ON labeling technique. KOD and Vent(exo-) DNA polymerases, belonging to the B family, tolerate all the three modified nucleotides and produce ONs with multiple labels. Importantly, the benzofuran-modified nucleotide (BFdUTP) serves as an excellent reporter by providing real-time fluorescence readouts to monitor enzyme activity and estimate the binding events in the catalytic cycle. Further, a direct comparison of the incorporation profiles, fluorescence data, and crystal structure of a ternary complex of KlenTaq DNA polymerase with BFdUTP poised for catalysis provides a detailed understanding of the mechanism of incorporation of heterocycle-modified nucleotides.

DNA polymerases catalyze DNA synthesis during the replication, repair, and recombination of DNA. Based on phylogenetic analysis and primary protein sequences, DNA polymerases have been categorized into seven families: A, B, C, D, X, Y, and RT. This review presents generalized data on the catalytic mechanism of action of DNA polymerases. The structural features of different DNA polymerase families are described in detail. The discussion highlights the kinetics and conformational dynamics of DNA polymerases from all known polymerase families during DNA synthesis.

Human DNA polymerases can bypass DNA lesions performing translesion synthesis (TLS), a mechanism of DNA damage tolerance. Tumor cells use this mechanism to survive lesions caused by specific chemotherapeutic agents, resulting in treatment relapse. Moreover, TLS polymerases are error-prone and, thus, can lead to mutagenesis, increasing the resistance potential of tumor cells. DNA polymerase eta (pol eta) - a key protein from this group - is responsible for protecting against sunlight-induced tumors. Xeroderma Pigmentosum Variant (XP-V) patients are deficient in pol eta activity, which leads to symptoms related to higher sensitivity and increased incidence of skin cancer. Temozolomide (TMZ) is a chemotherapeutic agent used in glioblastoma and melanoma treatment. TMZ damages cells' genomes, but little is known about the role of TLS in TMZ-induced DNA lesions. This work investigates the effects of TMZ treatment in human XP-V cells, which lack pol eta, and in its complemented counterpart (XP-V comp). Interestingly, TMZ reduces the viability of XP-V cells compared to TLS proficient control cells. Furthermore, XP-V cells treated with TMZ presented increased phosphorylation of H2AX, forming γH2AX, compared to control cells. However, cell cycle assays indicate that XP-V cells treated with TMZ replicate damaged DNA and pass-through S-phase, arresting in the G2/M-phase. DNA fiber assay also fails to show any specific effect of TMZ-induced DNA damage blocking DNA elongation in pol eta deficient cells. These results show that pol eta plays a role in protecting human cells from TMZ-induced DNA damage, but this can be different from its canonical TLS mechanism. The new role opens novel therapeutic possibilities of using pol eta as a target to improve the efficacy of TMZ-based therapies against cancer., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Rev1 is a translesion DNA synthesis (TLS) polymerase involved in the bypass of adducted-guanine bases and abasic sites during DNA replication. During damage bypass, Rev1 utilizes a protein-template mechanism of DNA synthesis, where the templating DNA base is evicted from the Rev1 active site and replaced by an arginine side chain that preferentially binds incoming dCTP. Here, we utilize X-ray crystallography and molecular dynamics simulations to obtain structural insight into the dCTP specificity of Rev1. We show the Rev1 R324 protein-template forms sub-optimal hydrogen bonds with incoming dTTP, dGTP, and dATP that prevents Rev1 from adopting a catalytically competent conformation. Additionally, we show the Rev1 R324 protein-template forms optimal hydrogen bonds with incoming rCTP. However, the incoming rCTP adopts an altered sugar pucker, which prevents the formation of a catalytically competent Rev1 active site. This work provides novel insight into the mechanisms for nucleotide discrimination by the TLS polymerase Rev1., (© 2022. The Author(s).)

DNA polymerase eta (Pol η) is a eukaryotic member of the Y-family of DNA polymerase involved in translesion DNA synthesis and genome mutagenesis. Recently, several translesion DNA synthesis polymerases have been found to function in repair of DNA double-strand breaks (DSBs). However, the role of Pol η in promoting DSB repair remains to be well defined. Here, we demonstrated that Pol η could be targeted to etoposide (ETO)-induced DSBs and that depletion of Pol η in cells causes increased sensitivity to ETO. Intriguingly, depletion of Pol η also led to a nonhomologous end joining repair defect in a catalytic activity-independent manner. We further identified the scaffold protein Kap1 as a novel interacting partner of Pol η, the depletion of which resulted in impaired formation of Pol η and Rad18 foci after ETO treatment. Additionally, overexpression of Kap1 failed to restore Pol η focus formation in Rad18-deficient cells after ETO treatment. Interestingly, we also found that Kap1 bound to Rad18 in a Pol η-dependent manner, and moreover, depletion of Kap1 led to a significant reduction in Rad18-Pol η association, indicating that Kap1 forms a ternary complex with Rad18 and Pol η to stabilize Rad18-Pol η association. Our findings demonstrate that Kap1 could regulate the role of Pol η in ETO-induced DSB repair via facilitating Rad18 recruitment and stabilizing Rad18-Pol η association., Competing Interests: Conflict of interest statement The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Error-free replication of DNA is essential for life. Despite the proofreading capability of several polymerases, intrinsic polymerase fidelity is in general much higher than what base-pairing energies can provide. Although researchers have investigated this long-standing question with kinetics, structural determination, and computational simulations, the structural factors that dictate polymerase fidelity are not fully resolved. Time-resolved crystallography has elucidated correct nucleotide incorporation and established a three-metal-ion-dependent catalytic mechanism for polymerases. Using X-ray time-resolved crystallography, we visualize the complete DNA misincorporation process catalyzed by DNA polymerase η. The resulting molecular snapshots suggest primer 3´-OH alignment mediated by A-site metal ion binding is the key step in substrate discrimination. Moreover, we observe that C-site metal ion binding preceded the nucleotidyl transfer reaction and demonstrate that the C-site metal ion is strictly required for misincorporation. Our results highlight the essential but separate roles of the three metal ions in DNA synthesis., (© 2022. The Author(s).)

Most insertions or deletions generated by CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9) endonucleases are short (<25 bp), but unpredictable on-target long DNA deletions (>500 bp) can be observed. The possibility of generating long on-target DNA deletions poses safety risks to somatic genome editing and makes the outcomes of genome editing less predictable. Methods for generating refined mutations are desirable but currently unavailable. Here, we show that fusing Escherichia coli DNA polymerase I or the Klenow fragment to Cas9 greatly increases the frequencies of 1-bp deletions and decreases >1-bp deletions or insertions. Importantly, doing so also greatly decreases the generation of long deletions, including those >2 kb. In addition, templated insertions (the insertion of the nucleotide 4 nt upstream of the protospacer adjacent motif) were increased relative to other insertions. Counteracting DNA resection was one of the mechanisms perturbing deletion sizes. Targeting DNA polymerase to double-strand breaks did not increase off-targets or base substitution rates around the cleavage sites, yet increased editing efficiency in primary cells. Our strategy makes it possible to generate refined DNA mutations for improved safety without sacrificing efficiency of genome editing., (© The Author(s) 2022. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Translesion synthesis (TLS) by specialized DNA polymerases (Pols) is an evolutionarily conserved mechanism for tolerating replication-blocking DNA lesions. Using the Escherichia coli dinB -encoded Pol IV as a model to understand how TLS is coordinated with the actions of the high-fidelity Pol III replicase, we previously described a novel Pol IV mutant containing a threonine 120-to-proline mutation (Pol IV-T120P) that failed to exchange places with Pol III at the replication fork in vitro as part of a Pol III-Pol IV switch. This in vitro defect correlated with the inability of Pol IV-T120P to support TLS in vivo , suggesting Pol IV gains access to the DNA, at least in part, via a Pol III-Pol IV switch. Interaction of Pol IV with the β sliding clamp and the single-stranded DNA binding protein (SSB) significantly stimulates Pol IV replication and facilitates its access to the DNA. In this work, we demonstrate that Pol IV interacts physically with Pol III. We further show that Pol IV-T120P interacts normally with the β clamp, but is impaired in interactions with the α catalytic and εθ proofreading subunits of Pol III, as well as SSB. Taken together with published work, these results provide strong support for the model in which Pol IV-Pol III and Pol IV-SSB interactions help to regulate the access of Pol IV to the DNA. Finally, we describe several additional E. coli Pol-Pol interactions, suggesting Pol-Pol interactions play fundamental roles in coordinating bacterial DNA replication, DNA repair, and TLS. IMPORTANCE Specialized DNA polymerases (Pols) capable of catalyzing translesion synthesis (TLS) generate mutations that contribute to bacterial virulence, pathoadaptation, and antimicrobial resistance. One mechanism by which the bacterial TLS Pol IV gains access to the DNA to generate mutations is by exchanging places with the bacterial Pol III replicase via a Pol III-Pol IV switch. Here, we describe multiple Pol III-Pol IV interactions and discuss evidence that these interactions are required for the Pol III-Pol IV switch. Furthermore, we describe several additional E. coli Pol-Pol interactions that may play fundamental roles in managing the actions of the different bacterial Pols in DNA replication, DNA repair, and TLS.

The charge states of proteins can greatly influence their stabilities and interactions with substrates, and the addition of multiple charges (supercharging) has been shown to be a successful approach for engineering protein stability and function. The addition of a fast-folding fusion domain to the Bacillus stearothermophilus DNA polymerase improved its functionality in isothermal amplification assays, and further charge engineering of this domain has increased both protein stability and diagnostics performance. When combined with mutations that stabilize the core of the protein, the charge-engineered fusion domain leads to the ability to carry out loop-mediated isothermal amplification (LAMP) at temperatures up to 74° C or in the presence of high concentrations of urea, with detection times under 10 min. Adding both positive and negative charges to the fusion domain led to changes in the relative reverse transcriptase and DNA polymerase activities of the polymerase. Overall, the development of a modular fusion domain whose charged surface can be modified at will should prove to be of use in the engineering of other polymerases and, in general, may prove useful for protein stabilization.

Non-enzymatic alkylation on DNA often generates N7-alkyl-2'-deoxyguanosine (N7alkylG) adducts as major lesions. N7alkylG adducts significantly block replicative DNA polymerases and can be bypassed by translesion synthesis (TLS) polymerases such as polymerase η (polη). To gain insights into the bypass of N7alkylG by TLS polymerases, we conducted kinetic and structural studies of polη catalyzing across N7BnG, a genotoxic lesion generated by the carcinogenic N -nitrosobenzylmethylamine. The presence of templating N7BnG in the polη catalytic site decreased the replication fidelity by ∼9-fold, highlighting the promutagenicity of N7BnG. The catalytic efficiency for dCTP incorporation opposite N7BnG decreased ∼22-fold and ∼7-fold compared to the incorporation opposite undamaged guanine in the presence of Mg 2+ and Mn 2+ , respectively. A crystal structure of the complexes grown with polη, templating N7BnG, incoming dCTP, and Mg 2+ ions showed the lack of the incoming nucleotide and metal cofactors in the polη catalytic site. Interestingly, the templating N7BnG adopted a syn conformation, which has not been observed in the published N7alkylG structures. The preferential formation of syn- N7BnG conformation at the templating site may deter the binding of an incoming dCTP, causing the inefficient bypass by polη. In contrast, the use of Mn 2+ in place of Mg 2+ in co-crystallization yielded a ternary complex displaying an anti -N7BnG:dCTP base pair and catalytic metal ions, which would be a close mimic of a catalytically competent state. We conclude that certain bulky N7-alkylG lesions can slow TLS polymerase-mediated bypass by adopting a catalytically unfavorable syn conformation in the replicating base pair site.

Xeroderma pigmentosum (XP) is a rare autosomal recessive genetic disorder characterized by severe sensitivity of skin to sunlight and an increased risk of skin cancer. XP variant (XPV), a milder subtype, is caused by variants in the POLH gene. POLH encodes an error-prone DNA-polymerase eta (pol eta) which performs translesion synthesis past ultraviolet photoproducts. The current study documents the clinical and genetic investigations of two large consanguineous Pakistani families affected with XPV. In family 1, whole exome sequencing (WES) revealed a novel frameshift variant, c.1723dupG (p.(Val575Glyfs*4)), of POLH , which is predicted to cause frameshift and premature truncation of the encoded enzyme. Indeed, our ex vivo studies in HEK293T cells confirmed the truncation of the encoded protein due to the c.1723dupG variant. In family 2, Sanger sequencing of POLH exons, revealed a recurrent nonsense variant, c.437dupA (p.Tyr146*). POLH forms a hetero-tetrameric POLZ complex with REV3L, REV7, POLD2 and POLD3. Next, we performed in silico analysis of POLH and other POLZ complex genes expression in publicly available single cell mRNAseq datasets from adult human healthy and aging skin. We found overlapping expression of POLH, REV3L and POLD2 in multiple cell types including differentiated and undifferentiated keratinocytes, pericytes and melanocytes in healthy skin. However, in aging human skin, POLH expression is reduced in compare to its POLZ complex partners. Insights from our study will facilitate counseling regarding the molecular and phenotypic landscape of POLH -related XPV.

The tail of replication-dependent histone H3.1 varies from that of replication-independent H3.3 at the amino acid located at position 31 in plants and animals, but no function has been assigned to this residue to demonstrate a unique and conserved role for H3.1 during replication. We found that TONSOKU (TSK/TONSL), which rescues broken replication forks, specifically interacts with H3.1 via recognition of alanine 31 by its tetratricopeptide repeat domain. Our results indicate that genomic instability in the absence of ATXR5/ATXR6-catalyzed histone H3 lysine 27 monomethylation in plants depends on H3.1, TSK, and DNA polymerase theta (Pol θ). This work reveals an H3.1-specific function during replication and a common strategy used in multicellular eukaryotes for regulating post-replicative chromatin maturation and TSK, which relies on histone monomethyltransferases and reading of the H3.1 variant.

B-family DNA polymerases, which are found in eukaryotes, archaea, viruses, and some bacteria, participate in DNA replication and repair. Starting from the N-terminus of archaeal and bacterial B-family DNA polymerases, three domains include the N-terminal, exonuclease, and polymerase domains. The N-terminal domain of the archaeal B-family DNA polymerase has a conserved deoxyuracil-binding pocket for specially binding the deoxyuracil base on DNA. The exonuclease domain is responsible for removing the mismatched base pair. The polymerase domain is the core functional domain and takes a highly conserved structure composed of fingers, palm and thumb subdomains. Previous studies have demonstrated that the thumb subdomain mainly functions as a DNA-binding element and has coordination with the exonuclease domain and palm subdomain. To further elucidate the possible functions of the thumb subdomain of archaeal B-family DNA polymerases, the thumb subdomain of Pyrococcus furiosus DNA polymerase was mutated, and the effects on three activities were characterized. Our results demonstrate that the thumb subdomain participates in the three activities of archaeal B-family DNA polymerases as a common structural element. Both the N-terminal deoxyuracil-binding pocket and thumb subdomain are critical for deoxyuracil binding. Moreover, the thumb subdomain assists DNA polymerization and hydrolysis reactions, but it does not contribute to the fidelity of DNA polymerization., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

The spontaneous depurination of genomic DNA occurs frequently and generates apurinic/pyrimidinic (AP) site damage that is mutagenic or lethal to cells. Error-prone DNA polymerases are specifically responsible for the translesion synthesis (TLS) of specific DNA damage, such as AP site damage, generally with relatively low fidelity. The Y-family DNA polymerases are the main error-prone DNA polymerases, and they employ three mechanisms to perform TLS, including template-skipping, dNTP-stabilized misalignment, and misincorporation-misalignment. The bypass mechanism of the dinB homolog (Dbh), an archaeal Y-family DNA polymerase from Sulfolobus acidocaldarius , is unclear and needs to be confirmed. In this study, we show that the Dbh primarily uses template skipping accompanied by dNTP-stabilized misalignment to bypass AP site analogs, and the incorporation of the first nucleotide across the AP site is the most difficult. Furthermore, based on the reported crystal structures, we confirmed that three conserved residues (Y249, R333, and I295) in the little finger (LF) domain and residue K78 in the palm subdomain of the catalytic core domain are very important for TLS. These results deepen our understanding of how archaeal Y-family DNA polymerases deal with intracellular AP site damage and provide a biochemical basis for elucidating the intracellular function of these polymerases.

DNA damage responses compete for cellular resources with metabolic pathways, but little is known about the metabolic consequences of impaired DNA replication, a process called replication stress. Here we characterized the metabolic consequences of DNA replication stress at endogenous DNA lesions by using mice with a disruption of Rev1, a translesion DNA polymerase specialized in the mutagenic replication of damaged DNA. Male and female Rev1 knockout (KO) mice were compared with wild-type (WT) mice and followed over time to study the natural course of body weight gain and glucose tolerance. Follow-up measurements were performed in female mice for in-depth metabolic characterization. Body weight and fat mass were only increased in female KO mice versus WT mice, whereas glucose intolerance and a reduction in lean mass were observed in both sexes. Female KO mice showed reduced locomotor activity while male KO mice showed increased activity as compared with their WT littermates. Further characterization of female mice revealed that lipid handling was unaffected by Rev1 deletion. An increased respiratory exchange ratio, combined with elevated plasma lactate levels and increased hepatic gluconeogenesis indicated problems with aerobic oxidation and increased reliance on anaerobic glycolysis. Supplementation with the NAD + precursor nicotinamide riboside to stimulate aerobic respiration failed to restore the metabolic phenotype. In conclusion, replication stress at endogenous DNA lesions induces a complex metabolic phenotype, most likely initiated by muscular metabolic dysfunction and increased dependence on anaerobic glycolysis. Nicotinamide riboside supplementation after the onset of the metabolic impairment did not rescue this phenotype. NEW & NOTEWORTHY An increasing number of DNA lesions interferes with cellular replication leading to metabolic inflexibility. We utilized Rev1 knockout mice as a model for replication stress, and show a sex-dependent metabolic phenotype, with a pronounced reduction of lean mass and glucose tolerance. These data indicate that in obesity, we may end up in an infinite loop where metabolic disturbance promotes the formation of DNA lesions, which in turn interferes with cellular replication causing further metabolic disturbances.

Faithful replication of genomic DNA by high-fidelity DNA polymerases is crucial for the survival of most living organisms. While high-fidelity DNA polymerases favor canonical base pairs over mismatches by a factor of ∼1 × 10 5 , fidelity is further enhanced several orders of magnitude by a 3'-5' proofreading exonuclease that selectively removes mispaired bases in the primer strand. Despite the importance of proofreading to maintaining genome stability, it remains much less studied than the fidelity mechanisms employed at the polymerase active site. Here we characterize the substrate specificity for the proofreading exonuclease of a high-fidelity DNA polymerase by investigating the proofreading kinetics on various DNA substrates. The contribution of the exonuclease to net fidelity is a function of the kinetic partitioning between extension and excision. We show that while proofreading of a terminal mismatch is efficient, proofreading a mismatch buried by one or two correct bases is even more efficient. Because the polymerase stalls after incorporation of a mismatch and after incorporation of one or two correct bases on top of a mismatch, the net contribution of the exonuclease is a function of multiple opportunities to correct mistakes. We also characterize the exonuclease stereospecificity using phosphorothioate-modified DNA, provide a homology model for the DNA primer strand in the exonuclease active site, and propose a dynamic structural model for the transfer of DNA from the polymerase to the exonuclease active site based on MD simulations., Competing Interests: Conflict of interest K. A. J. is president of KinTek Corporation, which provided the RQF-3 rapid quench flow instrument and KinTek Explorer software used in this study., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

The B-family multi-subunit DNA polymerase ζ (Polζ) is important for translesion DNA synthesis (TLS) during replication, due to its ability to extend synthesis past nucleotides opposite DNA lesions and mismatched base pairs. We present a cryo-EM structure of Saccharomyces cerevisiae Polζ with an A:C mismatch at the primer terminus. The structure shows how the Polζ active site responds to the mismatched duplex DNA distortion, including the loosening of key protein-DNA interactions and a fingers domain in an "open" conformation, while the incoming dCTP is still able to bind for the extension reaction. The structure of the mismatched DNA-Polζ ternary complex reveals insights into mechanisms that either stall or favor continued DNA synthesis in eukaryotes., (© 2022. The Author(s).)

Apurinic/apyrimidinic (AP) sites appear in DNA spontaneously and as intermediates of base excision DNA repair. AP sites are noninstructive lesions: they strongly block DNA polymerases, and if bypassed, the nature of the incorporated dNMP is mostly guided by the interactions within the polymerase-DNA active site. Many DNA polymerases follow the "A-rule", preferentially incorporating dAMP opposite to natural AP sites. Methoxyamine (MX), a small molecule, efficiently reacts with the aldehyde moiety of natural AP sites, thereby preventing their cleavage by APEX1, the major human AP endonuclease. MX is currently regarded as a possible sensitizer of cancer cells toward DNA-damaging drugs. To evaluate the mutagenic potential of MX, we have studied the utilization of various dNTPs by five DNA polymerases of different families encountering MX-AP adducts in the template in comparison with the natural aldehydic AP site. The Klenow fragment of Escherichia coli DNA polymerase I strictly followed the A-rule with both natural AP and MX-adducted AP sites. Phage RB69 DNA polymerase, a close relative of human DNA polymerases δ and ε, efficiently incorporated both dAMP and dGMP. DNA polymerase β mostly incorporated dAMP and dCMP, preferring dCMP opposite to the natural AP site and dAMP opposite to the MX-AP site, while DNA polymerase λ was selective for dGMP, apparently via the primer misalignment mechanism. Finally, translesion DNA polymerase κ also followed the A-rule for MX-AP and additionally incorporated dCMP opposite to a natural AP site. Overall, the MX-AP site, despite structural differences, was similar to the natural AP site in terms of the dNMP misincorporation preference but was bypassed less efficiently by all polymerases except for Pol κ.

Sliding clamps are ring-shaped protein complexes that are integral to the DNA replication machinery of all life. Sliding clamps are opened and installed onto DNA by clamp loader AAA+ ATPase complexes. However, how a clamp loader opens and closes the sliding clamp around DNA is still unknown. Here, we describe structures of the Saccharomyces cerevisiae clamp loader Replication Factor C (RFC) bound to its cognate sliding clamp Proliferating Cell Nuclear Antigen (PCNA) en route to successful loading. RFC first binds to PCNA in a dynamic, closed conformation that blocks both ATPase activity and DNA binding. RFC then opens the PCNA ring through a large-scale 'crab-claw' expansion of both RFC and PCNA that explains how RFC prefers initial binding of PCNA over DNA. Next, the open RFC:PCNA complex binds DNA and interrogates the primer-template junction using a surprising base-flipping mechanism. Our structures indicate that initial PCNA opening and subsequent closure around DNA do not require ATP hydrolysis, but are driven by binding energy. ATP hydrolysis, which is necessary for RFC release, is triggered by interactions with both PCNA and DNA, explaining RFC's switch-like ATPase activity. Our work reveals how a AAA+ machine undergoes dramatic conformational changes for achieving binding preference and substrate remodeling., Competing Interests: CG, XL, JP, NS, JH, GD, BK No competing interests declared, (© 2022, Gaubitz et al.)

A modified SELEX (Systematic Evolution of Ligands by Exponential Enrichment) pr,otocol (referred to as PT SELEX) was used to select primer-template (P/T) sequences that bound to the vaccinia virus polymerase catalytic subunit (E9) with enhanced affinity. A single selected P/T sequence (referred to as E9-R5-12) bound in physiological salt conditions with an apparent equilibrium dissociation constant (K D,app ) of 93 ± 7 nM. The dissociation rate constant ( k off ) and binding half-life (t 1/2 ) for E9-R5-12 were 0.083 ± 0.019 min -1 and 8.6 ± 2.0 min, respectively. The values indicated a several-fold greater binding ability compared to controls, which bound too weakly to be accurately measured under the conditions employed. Loop-back DNA constructs with 3'-recessed termini derived from E9-R5-12 also showed enhanced binding when the hybrid region was 21 nucleotides or more. Although the sequence of E9-R5-12 matched perfectly over a 12-base-pair segment in the coding region of the virus B20 protein, there was no clear indication that this sequence plays any role in vaccinia virus biology, or a clear reason why it promotes stronger binding to E9. In addition to E9, five other polymerases (HIV-1, Moloney murine leukemia virus, and avian myeloblastosis virus reverse transcriptases (RTs), and Taq and Klenow DNA polymerases) have demonstrated strong sequence binding preferences for P/Ts and, in those cases, there was biological or potential evolutionary relevance. For the HIV-1 RT, sequence preferences were used to aid crystallization and study viral inhibitors. The results suggest that several other DNA polymerases may have P/T sequence preferences that could potentially be exploited in various protocols.

Translesion DNA synthesis (TLS) is a fundamental damage bypass pathway that utilises specialised polymerases with relaxed template specificity to achieve replication through damaged DNA. Misinsertions by low fidelity TLS polymerases may introduce additional mutations on undamaged DNA near the original lesion site, which we termed collateral mutations. In this study, we used whole genome sequencing datasets of chicken DT40 and several human cell lines to obtain evidence for collateral mutagenesis in higher eukaryotes. We found that cisplatin and UVC radiation frequently induce close mutation pairs within 25 base pairs that consist of an adduct-associated primary and a downstream collateral mutation, and genetically linked their formation to TLS activity involving PCNA ubiquitylation and polymerase κ. PCNA ubiquitylation was also indispensable for close mutation pairs observed amongst spontaneously arising base substitutions in cell lines with disrupted homologous recombination. Collateral mutation pairs were also found in melanoma genomes with evidence of UV exposure. We showed that collateral mutations frequently copy the upstream base, and extracted a base substitution signature that describes collateral mutagenesis in the presented dataset regardless of the primary mutagenic process. Using this mutation signature, we showed that collateral mutagenesis creates approximately 10-20% of non-paired substitutions as well, underscoring the importance of the process., Competing Interests: The authors have declared that no competing interests exist.