Pole Research Articles

Development of a simple allele-specific PCR for the detection of pathogenic Vibrio cholerae O1 and O139 in seafood

This study developed a simple allele specific (AS) PCR assay for the detection of pathogenic Vibrio cholerae strains in seafood. A pair of primers, (1) an AS primer targeting the mutation at site G450 in the hapR2 allele and a (2) common forward primer, were designed to detect the pathogenic O1 and O139 strains. An artificial mismatch was also added to the AS primer to increase its specificity to the target allele. The PCR assays using different DNA polymerases of varying efficiencies were then optimized. The application of the AS primers in both conventional and real-time PCR assays was facilitated with enriched seafood samples spiked with known concentration of pathogenic V. cholerae. The designed AS primers showed high specificity and sensitivity to the hapR2 allele for both conventional and real-time PCR assays. Enrichment of seafood samples for 8 h is recommended to detect viable pathogenic V. cholerae presence in seafood. In this study, we developed an AS-PCR that is simple, rapid, and a low-cost alternative for routine detection of pathogenic V. cholerae in different seafood samples thus aiding in the surveillance and implementation of food safety measures at the seafood post-harvest level.

DNA Polymerase Locks Replication Fork Under Stress.

Replication of DNA requires the parental DNA to be unwound to allow the genetic information to be faithfully duplicated by the replisome. While this function is usually shared by a host of proteins in the replisome, notably DNA polymerase (DNAP) and helicase, the consequence of DNAP synthesizing DNA while decoupled from helicase remains not well understood. The unwinding of downstream DNA poses significant stress to DNAP, and the interaction between DNAP and the replication fork may affect replication restart. In this work, we examined the consequences of DNAP working against the stress of the DNA replication fork. We found that prolonged exposure of DNAP to the stress of the replication fork inactivates replication. Surprisingly, replication inactivation was often accompanied by a strong DNAP interaction with the leading and lagging strands at the fork, locking the fork in place. We demonstrated that fork locking is a consequence of DNAP forward translocation, and the exonuclease activity of DNAP, which allows DNAP to move in reverse, is essential in protecting the fork from inactivation. Furthermore, we found the locking configuration is not reversible by the subsequent addition of helicase. Collectively, this study provides a deeper understanding of the DNAP-fork interaction and mechanism in keeping the replication fork active during replication stress.

Human DNA polymerase ε is a source of C>T mutations at CpG dinucleotides

C-to-T transitions in CpG dinucleotides are the most prevalent mutations in human cancers and genetic diseases. These mutations have been attributed to deamination of 5-methylcytosine (5mC), an epigenetic modification found on CpGs. We recently linked CpG>TpG mutations to replication and hypothesized that errors introduced by polymerase ε (Pol ε) may represent an alternative source of mutations. Here we present a new method called polymerase error rate sequencing (PER-seq) to measure the error spectrum of DNA polymerases in isolation. We find that the most common human cancer-associated Pol ε mutant (P286R) produces an excess of CpG>TpG errors, phenocopying the mutation spectrum of tumors carrying this mutation and deficiencies in mismatch repair. Notably, we also discover that wild-type Pol ε has a sevenfold higher error rate when replicating 5mCpG compared to C in other contexts. Together, our results from PER-seq and human cancers demonstrate that replication errors are a major contributor to CpG>TpG mutagenesis in replicating cells, fundamentally changing our understanding of this important disease-causing mutational mechanism.

Template-Independent Enzymatic RNA Synthesis.

A route to prepare ribonucleoside triphosphates featuring a 3'-aminoxy (3'-O-NH 2 ) removable blocking group is reported here. We then show that versions of two DNA polymerases, human DNA polymerase theta (Polθ) and mimiviral PrimPol, accept these triphosphates as substrates to add single nucleotides to an RNA primer under engineered conditions. Cleaving the O-N bond in the 3'-O-NH 2 group within the extended primer regenerates the 3'-OH group, facilitating subsequent polymerase cycles that add a second, selected, nucleotide. These enzymes and triphosphates together enable template-independent enzymatic RNA synthesis (TIERS) exploiting a cyclic reversible termination framework. The study shows that this process is ready for instrument adaptation by using it to add three ribonucleotides in three cycles using an engineered Polθ. This work creates a new way to synthesize RNA with a de novo defined sequence, without requiring the protecting groups, hazardous solvents, and sensitive reagents that bedevil phosphoramidite-based RNA synthesis.

DNA polymerase ε produces elevated C-to-T mutations at methylated CpG dinucleotides.

DNA polymerase ε produces elevated C-to-T mutations at methylated CpG dinucleotides.

Navigating directed evolution efficiently: optimizing selection conditions and selection output analysis.

Directed evolution is a powerful tool that can bypass gaps in our understanding of the sequence-function relationship of proteins and still isolate variants with desired activities, properties, and substrate specificities. The rise of directed evolution platforms for polymerase engineering has accelerated the isolation of xenobiotic nucleic acid (XNA) synthetases and reverse transcriptases capable of processing a wide array of unnatural XNAs which have numerous therapeutic and biotechnological applications. Still, the current generation of XNA polymerases functions with significantly lower efficiency than the natural counterparts and retains a significant level of DNA polymerase activity which limits their in vivo applications. Although directed evolution approaches are continuously being developed and implemented to improve XNA polymerase engineering, the field lacks an in-depth analysis of the effect of selection parameters, library construction biases and sampling biases. Focusing on the directed evolution pipeline for DNA and XNA polymerase engineering, this work sets out a method for understanding the impact of selection conditions on selection success and efficiency. We also explore the influence of selection conditions on fidelity at the population and individual mutant level. Additionally, we explore the sequencing coverage requirements in directed evolution experiments, which differ from genome assembly and other -omics approaches. This analysis allowed us to identify the sequencing coverage threshold for the accurate and precise identification of significantly enriched mutants. Overall, this study introduces a robust methodology for optimizing selection protocols, which effectively streamlines selection processes by employing small libraries and cost-effective NGS sequencing. It provides valuable insights into critical considerations, thereby enhancing the overall effectiveness and efficiency of directed evolution strategies applicable to enzymes other than the ones considered here.

Engineering Phi29-DNAP Variants for Customized DNA Hydrogel Materials.

DNA hydrogels, which hold potential for use in medicine, biosensors, and tissue engineering, can be produced through enzymatic rolling circle amplification (RCA) using phi29 DNA polymerase (DNAP). This paper introduces new DNAP variants designed for RCA-based DNA hydrogel production, featuring enzymes with modified DNA binding, enhanced thermostability, reduced exonuclease activity, and protein tags for fluorescence detection or specific immobilization. We evaluated these enzymes by quantifying DNA output via quantitative PCR (qPCR) and assessing hydrogel mechanical properties through micromechanical indentation. The results showed that most variants generated similar DNA amounts and hydrogels with comparable mechanical properties. Additionally, all variants successfully incorporated non-natural nucleotides, such as base-modified dGTP derivatives and 2'-fluoro-dGTP, during RCA. This study's robust analytical approach offers a strong foundation for selecting new enzymes and producing DNA hydrogels with tailored material properties.

Histone-like transcription factor Hfl1p in Candida albicans harmonizes nuclear and mitochondrial genomic network in regulation of energy metabolism and filamentation development

ABSTRACT Candida albicans is an opportunistic fungal pathogen known for surviving in various nutrient-limited conditions within the host and causing infections. Our prior research revealed that Hfl1p, an archaeal histone-like or Hap5-like protein, is linked to mitochondrial ATP generation and yeast-hyphae morphogenesis. However, the specific roles of Hfl1p in these virulence behaviours, through its function in the CBF/NF-Y complex or as a DNA polymerase II subunit, remain unclear. This study explores Hfl1p’s diverse functions in energy metabolism and morphogenesis. By combining proteomic analysis and phenotypic evaluations of the hfl1Δ/hfl1Δ mutant with ChIP data, we found that Hfl1p significantly impacts mitochondrial DNA-encoded CI subunits, the tricarboxylic acid (TCA) cycle, and morphogenetic pathways. This influence occurs either independently or alongside other transcription factors recognizing a conserved DNA motif (TAXXTAATTA). These findings emphasize Hfl1p’s critical role in linking carbon metabolism and mitochondrial respiration to the yeast-to-filamentous form transition, enhancing our understanding of C. albicans’ metabolic adaptability during morphological transition, an important pathogenic trait of this fungus. This could help identify therapeutic targets by disrupting the relationship between energy metabolism and cell morphology in C. albicans.

Optimized Method for the Synthesis of Alkyne-Modified 2'-Deoxynucleoside Triphosphates.

A general approach is presented for synthesizing alkyne-modified nucleoside triphosphates via the Sonogashira cross-coupling reaction of unprotected halogenated 2'-deoxynucleoside, followed by monophosphorylation and the reaction of the corresponding phosphoromorpholidate with tributylammonium pyrophosphate. A highly efficient approach for the milligram-scale synthesis of base-modified nucleoside triphosphates with an amino acid-like side chain was developed. The present chemical method outweighs the other reported methods of a base-modified nucleoside triphosphates synthesis in terms of it being a protection-free strategy, the shortening of reaction steps, and increased yields (about 70%). The resulting 8-alkynylated dATP was tested as a substrate for DNA polymerases in a primer extension reaction.

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).

Remdesivir triphosphate is a valid substrate to initiate synthesis of DNA primers by human PrimPol

Remdesivir is a broad-spectrum antiviral drug which has been approved to treat COVID-19. Remdesivir is in fact a prodrug, which is metabolized in vivo into the active form remdesivir triphosphate (RTP), an analogue of adenosine triphosphate (ATP) with a cyano group substitution in the carbon 1’ of the ribose (1’-CN). RTP is a substrate for RNA synthesis and can be easily incorporated by viral RNA-dependent RNA polymerases (RdRp). Importantly, once remdesivir is incorporated (now monophosphate), it will act as a delayed chain terminator, thus blocking viral RNA synthesis. It has been reported that mitochondrial Polγ is also blocked in vitro by RTP, but the low impact in vivo on mitochondrial DNA replication stalling is likely due to repriming by the human DNA-directed DNA Primase/Polymerase (HsPrimPol), which also operates in mitochondria. In this work, we have tested if RTP is a valid substrate for both DNA primase and DNA polymerase activities of HsPrimPol, and its impact in the production of mature DNA primers. RTP resulted to be an invalid substrate for elongation, but it can be used to initiate primers at the 5´site, competing with ATP. Nevertheless, RTP-initiated primers are abortive, ocassionally reaching a maximal length of 4–5 nucleotides, and do not support elongation mediated by primer/template distortions. However, considering that the concentration of ATP, the natural substrate, is much higher than the intracellular concentration of RTP, it is unlikely that HsPrimPol would use RTP for primer synthesis during a remdesivir treatment in real patients.

To boldly go where no microRNAs have gone before: spaceflight impact on risk for small-for-gestational-age infants

In the era of renewed space exploration, comprehending the effects of the space environment on human health, particularly for deep space missions, is crucial. While extensive research exists on the impacts of spaceflight, there is a gap regarding female reproductive risks. We hypothesize that space stressors could have enduring effects on female health, potentially increasing risks for future pregnancies upon return to Earth, particularly related to small-for-gestational-age (SGA) fetuses. To address this, we identify a shared microRNA (miRNA) signature between SGA and the space environment, conserved across humans and mice. These miRNAs target genes and pathways relevant to diseases and development. Employing a machine learning approach, we identify potential FDA-approved drugs to mitigate these risks, including estrogen and progesterone receptor antagonists, vitamin D receptor antagonists, and DNA polymerase inhibitors. This study underscores potential pregnancy-related health risks for female astronauts and proposes pharmaceutical interventions to counteract the impact of space travel on female health.

Optimization of loop-mediated isothermal amplification assay for sunflower mildew disease detection

Loop-Mediated Isothermal Amplification (LAMP) represents a valuable technique for DNA/RNA detection, known for its exceptional sensitivity, specificity, speed, accuracy, and affordability. This study focused on optimizing a LAMP-based method to detect early signs of Plasmopara halstedii, the casual pathogen of sunflower downy mildew, a severe threat to sunflower crops. Specifically, a set of six LAMP primers (two outer, two inner, and two loop) were designed from P. halstedii genomic DNA, targeting the ribosomal Large Subunit (LSU). These primers were verified by in silico analysis and experimental validation using both target and non-target species' DNAs. Optimizations encompassing reaction conditions (temperature, time) and component concentrations (magnesium, Bst DNA polymerase, primers, and dNTP) were determined. Validation of these optimizations was performed by agarose gel electrophoresis. Furthermore, various colorimetric chemicals (Neutral Red, Hydroxynaphthol Blue, SYBR Safe, Thiazole Green) were evaluated to facilitate method analysis, and the real-time analysis has been optimized, presenting multiple approaches for detecting sunflower downy mildew using the LAMP technique. The analytical sensitivity of the method was confirmed by detecting P. halstedii DNA concentrations as low as 0.5 pg/μl. This pioneering study, establishing P. halstedii detection through the LAMP method, stands as unique in its field. The precision, robustness, and practicality of the LAMP protocol make it an ideal choice for studies focusing on sunflower mildew, emphasizing its recommended use due to its operational ease and reliability.

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.

B-237 Engineered Reverse Transcriptases and Taq DNA Polymerases deliver robust yield in the presence of clinically relevant inhibitors

Abstract Background The past five years has seen explosive growth in molecular diagnostic assays that rely on sensitive and accurate detection of nucleic acids across a wide range of sample types. As the assays are required to become increasingly sensitive, the need for highly functional and purpose-built enzymatic tools has grown. Reverse transcriptase (RT) and Taq DNA Polymerase (Taq DNAP) are commonly used to detect RNA and/or DNA viruses in patient samples for point of care (POC) diagnostics. However, crude patient samples introduce inhibitors that decrease enzyme activity and increase false negative results in POC testing. In addition, there is a need for faster turn-around time for patient diagnosis which can be enabled by engineering more active and sensitive Taq variants. Herein, we present on our engineered RT and Taq DNAP enzymes that were developed to confer greater activity, thermostability, inhibitor tolerance and/or sensitivity. Methods We take a 3-pronged approach to protein engineering: rational design, computational design and directed evolution. Using these techniques, we identified and characterized many RT and Taq DNAP variants. We deeply characterized each RT variant by measuring processivity, inhibitor tolerance and thermostability. First strand synthesis was done at temperatures from 42-65°C in the presence and absence of various clinically relevant inhibitors followed by qPCR to assess cDNA yield. Next, we characterized the inhibitor tolerance of the Taq variants by performing qPCR reactions in the presence of inhibitors. To determine if the Taq variant would be an appropriate fit for fast PCR, the polymerization activity of the Taq DNAP variants were characterized using a primer extension assay. Results Reverse transcriptases termed; StellarScript, StellarScript HT and StellarScript HT+ exhibit high processivity at 42°C. StellarScript HT+ exhibited the highest inhibitor tolerance, processivity and thermostability at temperatures up to 65°C thus making StellarScript HT+ well suited to produce high yields with clinical samples. Taq Pol variants were identified that efficiently amplify DNA in the presence of inhibitors. Conclusions Based on the increased polymerase activity and inhibitor tolerance, WMG Taq variants are well suited to give robust DNA yields with clinical samples. We were successful in engineering RT and Taq DNAP variants that are highly thermostable, active and inhibitor tolerant and thus will enable breakthroughs in existing POC diagnostics.

Microhomology-Mediated End-Joining Chronicles: Tracing the Evolutionary Footprints of Genome Protection.

The fidelity of genetic information is essential for cellular function and viability. DNA double-strand breaks (DSBs) pose a significant threat to genome integrity, necessitating efficient repair mechanisms. While the predominant repair strategies are usually accurate, paradoxically, error-prone pathways also exist. This review explores recent advances and our understanding of microhomology-mediated end joining (MMEJ), an intrinsically mutagenic DSB repair pathway conserved across organisms. Central to MMEJ is the activity of DNA polymerase theta (Polθ), a specialized polymerase that fuels MMEJ mutagenicity. We examine the molecular intricacies underlying MMEJ activity and discuss its function during mitosis, where the activity of Polθ emerges as a last-ditch effort to resolve persistent DSBs, especially when homologous recombination is compromised. We explore the promising therapeutic applications of targeting Polθ in cancer treatment and genome editing. Lastly, we discuss the evolutionary consequences of MMEJ, highlighting its delicate balance between protecting genome integrity and driving genomic diversity.

B-208 Room Temperature Stable PCR Reagents Preparation Using Capillary-Mediated Vitrification Process

Abstract Background Polymerase chain reaction (PCR) is a sensitive, and most used molecular technique for the disease diagnosis. Reagents used for PCR is available as a one-step and ready-to-use PCR Master Mix, which contains Taq DNA polymerase, dNTPs, MgC12, PCR reaction buffer. However, to preserve bioactivity, PCR reagents must be stored and distributed as frozen. Desiccation of PCR reagents can avoid cold storage and improper handling of wet reagents and its associated errors. We discovered capillary-mediated vitrification (CMV) processes can stabilize different biomolecules and allow them to be stored at ambient temperature. Here, we hypothesized that the CMV process can preserve and improve PCR reagents thermal stability, which will reduce PCR processing time and improve the testing throughput. Methods The CMV process was performed by mixing PCR Master Mix individual or all reagents such as Taq DNA polymerase or Hot Start Taq DNA polymerase, dNTPs, 16S rRNA primers, MgC12, and PCR reaction buffer with an excipient buffer and adding the mixture to a porous scaffold. The loaded scaffolds were placed into our vitrification chamber and dried for 30 min. After drying, the CMV-stabilized sample was tested or packed in Mylar pouches and were stored at ambient and elevated temperatures for 42 days. On the day of testing, the stabilized samples were eluted with nuclease-free water and E. coli genomic DNA was added, and the 50 uL reaction was tested by conventional PCR. The PCR product was visualized under agarose gel electrophoresis. Results To assess whether the CMV process maintains PCR reagents activity and not interfering with the assay, the individual PCR reagents and as a Master mix are preserved. The CMV stabilized polymerase enzyme, primer, or dNTPs amplified DNA similar to frozen reagents and produced comparable PCR products. Notably, CMV stabilized PCR Master Mix which contains either Taq DNA polymerase or Hot Start Taq DNA polymerase enzyme, dNTPs, primers, MgC12, and reaction buffer yielded intact amplified PCR product, which is comparable to frozen reagents. Likewise, a high intense PCR product was observed in the stabilized PCR Master Mix reagents when stored at room and elevated temperatures for 42 days. Conclusions The study results indicate that the CMV process stabilized and improved the PCR Master Mix reagents activity. The study suggests that the CMV technology has the potential to enable ambient storage and shipping of PCR reagents for molecular diagnostic and other related applications.

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.