Sequential requirements for distinct Polθ domains during theta-mediated end joining.

DNA polymerase θ (Polθ) plays a central role in a DNA double-strand break repair pathway termed theta-mediated end joining (TMEJ). TMEJ functions by pairing short-sequence "microhomologies" (MHs) in single-stranded DNA at each end of a break and subsequently initiating DNA synthesis. It is not known how the Polθ helicase domain (HD) and polymerase domain (PD) operate to bring together MHs and facilitate repair. To resolve these transient processes in real time, we utilized in vitro single-molecule FRET approaches and biochemical analyses. We find that the Polθ-HD mediates the initial capture of two ssDNA strands, bringing them in close proximity. The Polθ-PD binds and stabilizes pre-annealed MHs to form a synaptic complex (SC) and initiate repair synthesis. Individual synthesis reactions show that Polθ is inherently non-processive, accounting for complex mutational patterns during TMEJ. Binding of Polθ-PD to stem-loop-forming sequences can substantially limit synapsis, depending on the available dNTPs and sequence context.

Zebrafish polg2 knock-out recapitulates human POLG-disorders; implications for drug treatment.

The human mitochondrial DNA polymerase gamma is a holoenzyme, involved in mitochondrial DNA (mtDNA) replication and maintenance, composed of a catalytic subunit (POLG) and a dimeric accessory subunit (POLG2) conferring processivity. Mutations in POLG or POLG2 cause POLG-related diseases in humans, leading to a subset of Mendelian-inherited mitochondrial disorders characterized by mtDNA depletion (MDD) or accumulation of multiple deletions, presenting multi-organ defects and often leading to premature death at a young age. Considering the paucity of POLG2 models, we have generated a stable zebrafish polg2 mutant line (polg2ia304) by CRISPR/Cas9 technology, carrying a 10-nucleotide deletion with frameshift mutation and premature stop codon. Zebrafish polg2 homozygous mutants present slower development and decreased viability compared to wild type siblings, dying before the juvenile stage. Mutants display a set of POLG-related phenotypes comparable to the symptoms of human patients affected by POLG-related diseases, including remarkable MDD, altered mitochondrial network and dynamics, and reduced mitochondrial respiration. Histological analyses detected morphological alterations in high-energy demanding tissues, along with a significant disorganization of skeletal muscle fibres. Consistent with the last finding, locomotor assays highlighted a decreased larval motility. Of note, treatment with the Clofilium tosylate drug, previously shown to be effective in POLG models, could partially rescue MDD in Polg2 mutant animals. Altogether, our results point at zebrafish as an effective model to study the etiopathology of human POLG-related disorders linked to POLG2, and a suitable platform to screen the efficacy of POLG-directed drugs in POLG2-associated forms.

Recent advances in the synthesis of single-stranded DNA in vitro.

Single-stranded DNA (ssDNA) is the foundation of modern biology, with wide applications in gene editing, sequencing, DNA information storage, and materials science. However, synthesizing ssDNA with high efficiency, high throughput, and low error rate in vitro remains a major challenge. Various methods have been developed for ssDNA synthesis, and some significant results have been achieved. In this review, six main methods were introduced, including solid-phase oligonucleotide synthesis, terminal deoxynucleotidyl transferase-based ssDNA synthesis, reverse transcription, primer exchange reaction, asymmetric polymerase chain reaction, and rolling circle amplification. The advantages and limitations of each method were compared, as well as illustrate their representative achievements and applications. Especially, rolling circle amplification has received significant attention, including ssDNA synthesis, assembly, and application based on recent work. Finally, the future challenges and opportunities of ssDNA synthesis were summarized and discussed. Envisioning the development of new methods and significant progress will be made in the near future with the efforts of scientists around the world.

Neq2X7: a multi-purpose and open-source fusion DNA polymerase for advanced DNA engineering and diagnostics PCR.

Thermostable DNA polymerases, such as Taq isolated from the thermophilic bacterium Thermus aquaticus, enable one-pot exponential DNA amplification known as polymerase chain reaction (PCR). However, properties other than thermostability - such as fidelity, processivity, and compatibility with modified nucleotides - are important in contemporary molecular biology applications. Here, we describe the engineering and characterization of a fusion between a DNA polymerase identified in the marine archaea Nanoarchaeum equitans and a DNA binding domain from the thermophile Sulfolobus solfataricus. The fusion creates a highly active enzyme, Neq2X7, capable of amplifying long and GC-rich DNA, unaffected by replacing dTTP with dUTP in PCR, and tolerant to various known PCR inhibitors. This makes it an attractive DNA polymerase for use, e.g., with uracil excision (USER) DNA assembly and for contamination-free diagnostics. Using a magnification via nucleotide imbalance fidelity assay, Neq2X7 was estimated to have an error rate lower than 2 ∙ 10-5 bp-1 and an approximately 100x lower fidelity than the parental variant Neq2X, indicating a trade-off between fidelity and processivity - an observation that may be of importance for similarly engineered DNA polymerases. Neq2X7 is easy to produce for routine application in any molecular biology laboratory, and the expression plasmid is made freely available.

The Catalytic Activity of Human REV1 on Undamaged and Damaged DNA.

Eukaryotic REV1 serves as a scaffold protein for the coordination of DNA polymerases during DNA translesion synthesis. Besides this structural role, REV1 is a Y-family DNA polymerase with its own distributive deoxycytidyl transferase activity. However, data about the accuracy and efficiency of DNA synthesis by REV1 in the literature are contrasting. Here, we expressed and purified the full-length human REV1 from Saccharomyces cerevisiae and characterized its activity on undamaged DNA and a wide range of damaged DNA templates. We demonstrated that REV1 carried out accurate synthesis opposite 8-oxoG and O6-meG with moderate efficiency. It also replicated thymine glycol surprisingly well in an error-prone manner, but was blocked by the intrastrand 1,2-GG cisplatin crosslink. By using the 1,N6-ethenoadenine and 7-deaza-adenine lesions, we have provided biochemical evidence of the importance for REV1 functioning of the Hoogsteen face of template A, the second preferable template after G.

Expedient production of site specifically nucleobase-labelled or hypermodified RNA with engineered thermophilic DNA polymerases.

Innovative approaches to controlled nucleobase-modified RNA synthesis are urgently needed to support RNA biology exploration and to synthesize potential RNA therapeutics. Here we present a strategy for enzymatic construction of nucleobase-modified RNA based on primer-dependent engineered thermophilic DNA polymerases - SFM4-3 and TGK. We demonstrate introduction of one or several different base-modified nucleotides in one strand including hypermodified RNA containing all four modified nucleotides bearing four different substituents, as well as strategy for primer segment removal. We also show facile site-specific or segmented introduction of fluorophores or other functional groups at defined positions in variety of RNA molecules, including structured or long mRNA. Intriguing translation efficacy of single-site modified mRNAs underscores the necessity to study isolated modifications placed at designer positions to disentangle their biological effects and enable development of improved mRNA therapeutics. Our toolbox paves the way for more precise dissecting RNA structures and functions, as well as for construction of diverse types of base-functionalized RNA for therapeutic applications and diagnostics.

Discovery of a small-molecule inhibitor that traps Polθ on DNA and synergizes with PARP inhibitors.

The DNA damage response (DDR) protein DNA Polymerase θ (Polθ) is synthetic lethal with homologous recombination (HR) factors and is therefore a promising drug target in BRCA1/2 mutant cancers. We discover an allosteric Polθ inhibitor (Polθi) class with 4-6 nM IC50 that selectively kills HR-deficient cells and acts synergistically with PARP inhibitors (PARPi) in multiple genetic backgrounds. X-ray crystallography and biochemistry reveal that Polθi selectively inhibits Polθ polymerase (Polθ-pol) in the closed conformation on B-form DNA/DNA via an induced fit mechanism. In contrast, Polθi fails to inhibit Polθ-pol catalytic activity on A-form DNA/RNA in which the enzyme binds in the open configuration. Remarkably, Polθi binding to the Polθ-pol:DNA/DNA closed complex traps the polymerase on DNA for more than forty minutes which elucidates the inhibitory mechanism of action. These data reveal a unique small-molecule DNA polymerase:DNA trapping mechanism that induces synthetic lethality in HR-deficient cells and potentiates the activity of PARPi.

DNA polymerase λ Loop1 variant yields unexpected gain-of-function capabilities in nonhomologous end-joining.

DNA polymerases lambda (Polλ) and mu (Polµ) are X-Family polymerases that participate in DNA double-strand break (DSB) repair by the nonhomologous end-joining pathway (NHEJ). Both polymerases direct synthesis from one DSB end, using template derived from a second DSB end. In this way, they promote the NHEJ ligation step and minimize the sequence loss normally associated with this pathway. The two polymerases differ in cognate substrate, as Polλ is preferred when synthesis must be primed from a base-paired DSB end, while Polµ is required when synthesis must be primed from an unpaired DSB end. We generated a Polλ variant (PolλKGET) that retained canonical Polλ activity on a paired end-albeit with reduced incorporation fidelity. We recently discovered that the variant had unexpectedly acquired the activity previously unique to Polµ-synthesis from an unpaired primer terminus. Though the sidechains of the Loop1 region make no contact with the DNA substrate, PolλKGET Loop1 amino acid sequence is surprisingly essential for its unique activity during NHEJ. Taken together, these results underscore that the Loop1 region plays distinct roles in different Family X polymerases.

Influence of Nucleotide Context on Non-Specific Amplification of DNA with Bst exo- DNA Polymerase.

Isothermal nucleic acids amplification that requires DNA polymerases with strand-displacement activity gained more attention in the last two decades. Among the DNA polymerases with strand-displacement activity, Bst exo- is the most widely used. However, it tends to carry out nonspecific DNA synthesis through multimerization. In this study, the effect of nucleotide sequence on the Bst exo- binding with DNA and on the efficiency of multimerization initiation, are reported. Preference for binding of the "closed" form of Bst exo- to the purine-rich DNA sequences, especially those containing dG at the 3'-end of the growing chain was revealed using molecular docking of the single-stranded trinucleotides (sst) and trinucleotide duplexes (dst). The data obtained in silico were confirmed in the experiments using oligonucleotide templates that differ in the structure of the 3'- and 5'-terminal motifs. It has been shown that templates with the oligopurine 3'-terminal fragment and oligopyrimidine 5'-terminal part contribute to the earlier start of multimerization. The results can be used for design of nucleotide sequences suitable for reliable isothermal amplification. To avoid multimerization, DNA templates and primers containing terminal dA and/or dG nucleotides should be excluded.

Enzymatic Preparation of DNA with an Expanded Genetic Alphabet Using Terminal Deoxynucleotidyl Transferase and Its Applications.

Efficient preparation of DNA oligonucleotides containing unnatural nucleobases (UBs) that can pair with their cognates to form unnatural base pairs (UBPs) is an essential prerequisite for the application of UBPs in vitro and in vivo. Traditional preparation of oligonucleotides containing unnatural nucleobases largely relies on solid-phase synthesis, which needs to use unstable nucleoside phosphoramidites and a DNA synthesizer, and is environmentally unfriendly and limited in product length. To overcome these limitations of solid-phase synthesis, we developed enzymatic methods for daily laboratory preparation of DNA oligonucleotides containing unnatural nucleobase dNaM, dTPT3, or one of the functionalized dTPT3 derivatives, which can be used for orthogonal DNA labeling or the preparation of DNAs containing UBP dNaM-dTPT3, one of the most successful UBPs to date, based on the template-independent polymerase terminal deoxynucleotidyl transferase (TdT). Here, we first provide a detailed procedure for the TdT-based preparation of DNA oligonucleotides containing 3'-nucleotides of dNaM, dTPT3, or one of dTPT3 derivatives. We then present the procedures for enzyme-linked oligonucleotide assay (ELONA) and imaging of bacterial cells using DNA oligonucleotides containing 3'-nucleotides of dTPT3 derivatives with different functional groups. The procedure for enzymatic synthesis of DNAs containing an internal UBP dNaM-dTPT3 is also described. Hopefully, these methods will greatly facilitate the application of UBPs and the construction of semi-synthetic organisms with an expanded genetic alphabet.

HBV DNA polymerase upregulates the transcription of PD-L1 and suppresses T cell activity in hepatocellular carcinoma.

BACKGROUND: In HBV-associated HCC, T cells often exhibit a state of functional exhaustion, which prevents the immune response from rejecting the tumor and allows HCC to progress. Moreover, polymerase-specific T cells exhibit more severe T-cell exhaustion compared to core-specific T cells. However, whether HBV DNA polymerase drives HBV-specific CD8+ T cell exhaustion in HBV-related HCC remains unclear. METHODS: We constructed a Huh7 cell line stably expressing HA-HBV-DNA-Pol and applied co-culture systems to clarify its effect on immune cell function. We also examined how HBV-DNA-Pol modulated PD-L1 expression in HCC cells. In addition, HBV-DNA-Pol transgenic mice were used to elucidate the underlying mechanism of HBV-DNA-Pol/PD-L1 axis-induced T cell exhaustion. RESULTS: Biochemical analysis showed that Huh7 cells overexpressing HBV-DNA-Pol inhibited the proliferation, activation, and cytokine secretion of Jurkat cells and that this effect was dependent on their direct contact. A similar inhibitory effect was observed in an HCC mouse model. PD-L1 was brought to our attention during screening. Our results showed that the overexpression of HBV-DNA-Pol upregulated PD-L1 mRNA and protein expression. PD-L1 antibody blockade reversed the inhibitory effect of Huh7 cells overexpressing HBV-DNA-Pol on Jurkat cells. Mechanistically, HBV-DNA-Pol interacts with PARP1, thereby inhibiting the nuclear translocation of PARP1 and further upregulating PD-L1 expression. CONCLUSIONS: Our findings suggest that HBV-DNA-Pol can act as a regulator of PD-L1 in HCC, thereby directing anti-cancer immune evasion, which further provides a new idea for the clinical treatment of liver cancer.

Origin, evolution, and maintenance of gene-strand bias in bacteria.

Gene-strand bias is a characteristic feature of bacterial genome organization wherein genes are preferentially encoded on the leading strand of replication, promoting co-orientation of replication and transcription. This co-orientation bias has evolved to protect gene essentiality, expression, and genomic stability from the harmful effects of head-on replication-transcription collisions. However, the origin, variation, and maintenance of gene-strand bias remain elusive. Here, we reveal that the frequency of inversions that alter gene orientation exhibits large variation across bacterial populations and negatively correlates with gene-strand bias. The density, distance, and distribution of inverted repeats show a similar negative relationship with gene-strand bias explaining the heterogeneity in inversions. Importantly, these observations are broadly evident across the entire bacterial kingdom uncovering inversions and inverted repeats as primary factors underlying the variation in gene-strand bias and its maintenance. The distinct catalytic subunits of replicative DNA polymerase have co-evolved with gene-strand bias, suggesting a close link between replication and the origin of gene-strand bias. Congruently, inversion frequencies and inverted repeats vary among bacteria with different DNA polymerases. In summary, we propose that the nature of replication determines the fitness cost of replication-transcription collisions, establishing a selection gradient on gene-strand bias by fine-tuning DNA sequence repeats and, thereby, gene inversions.

A phosphamide nucleotide analog: a substrate for polymerase synthesis of DNA.

A type of modified nucleotide, deoxynucleotide γ-amidotriphosphates (dNTPγNH2s), exhibited around five times higher stability than dNTPs. These phosphamide nucleotides can be utilized by several DNA polymerases, and the amplification of a 10 kb DNA fragment through the polymerase chain reaction (PCR) can be accomplished even under conditions of high temperature, extended storage, or repeated freeze-thaw cycles. However, the control PCR with standard dNTPs was unsuccessful. These results indicate that dNTPγNH2s have the potential to substitute dNTPs in PCR.

WRN exonuclease imparts high fidelity on translesion synthesis by Y family DNA polymerases.

Purified translesion synthesis (TLS) DNA polymerases (Pols) replicate through DNA lesions with a low fidelity; however, TLS operates in a predominantly error-free manner in normal human cells. To explain this incongruity, here we determine whether Y family Pols, which play an eminent role in replication through a diversity of DNA lesions, are incorporated into a multiprotein ensemble and whether the intrinsically high error rate of the TLS Pol is ameliorated by the components in the ensemble. To this end, we provide evidence for an indispensable role of Werner syndrome protein (WRN) and WRN-interacting protein 1 (WRNIP1) in Rev1-dependent TLS by Y family Polη, Polι, or Polκ and show that WRN, WRNIP1, and Rev1 assemble together with Y family Pols in response to DNA damage. Importantly, we identify a crucial role of WRN's 3' → 5' exonuclease activity in imparting high fidelity on TLS by Y family Pols in human cells, as the Y family Pols that accomplish TLS in an error-free manner manifest high mutagenicity in the absence of WRN's exonuclease function. Thus, by enforcing high fidelity on TLS Pols, TLS mechanisms have been adapted to safeguard against genome instability and tumorigenesis.

Field-effect transistor biosensor with signal amplification by ternary initiation complexes for detection of wide-range RNA concentration.

Electrical detection of RNAs using transistor-based biosensors has attracted attention as a strategy for medical diagnosis and environmental monitoring. Herein, we demonstrated a proof-of-concept for specific, sensitive, and label-free RNA detection using a field-effect transistor (FET) biosensor with signal amplification by ternary initiation complexes (SATIC), which is an isothermal one-step nucleic acid amplification initiated by the combination of target RNA, circular DNA template and DNA primer. The SATIC system-applied FET biosensor specifically and quantitatively detected the target RNA with a single-nucleotide difference via the negative charges derived from the amplification products formed by a nucleic acid amplification reaction with φ29 DNA polymerase on the gate surface. In particular, the control of the amplification time allowed the detection of target RNA molecules over a wide concentration range, resulting in a detection limit of up to 6 copies/µL. Therefore, a transistor-based bioassay using the SATIC system could be useful for simple and sensitive nucleic acid analysis.

The conserved RNP motif of the herpes simplex virus 1 family B DNA polymerase is crucial for viral DNA synthesis but not polymerase activity.

The herpes simplex virus 1 DNA polymerase contains a highly conserved structural motif found in most family B polymerases and certain RNA-binding proteins. To investigate its importance within cells, we constructed a mutant virus with substitutions in two residues of the motif and a rescued derivative. The substitutions resulted in severe impairment of plaque formation, yields of infectious virus, and viral DNA synthesis while not meaningfully affecting expression of the mutant enzyme, its co-localization with the viral single-stranded DNA binding protein at intranuclear punctate sites in non-complementing cells or in replication compartments in complementing cells, or viral DNA polymerase activity. Taken together, our results indicate that the RNA binding motif plays a crucial role in herpes simplex virus 1 DNA synthesis through a mechanism separate from effects on polymerase activity, thus identifying a distinct essential function of this motif with implications for hypotheses regarding its biochemical functions.

Xanthomonas Phage PBR31: Classifying the Unclassifiable.

The ability of bacteriophages to destroy bacteria has made them the subject of extensive research. Interest in bacteriophages has recently increased due to the spread of drug-resistant bacteria, although genomic research has not kept pace with the growth of genomic data. Genomic analysis and, especially, the taxonomic description of bacteriophages are often difficult due to the peculiarities of the evolution of bacteriophages, which often includes the horizontal transfer of genes and genomic modules. The latter is particularly pronounced for temperate bacteriophages, which are capable of integration into the bacterial chromosome. Xanthomonas phage PBR31 is a temperate bacteriophage, which has been neither described nor classified previously, that infects the plant pathogen Xanthomonas campestris pv. campestris. Genomic analysis, including phylogenetic studies, indicated the separation of phage PBR31 from known classified bacteriophages, as well as its distant relationship with other temperate bacteriophages, including the Lederbervirus group. Bioinformatic analysis of proteins revealed distinctive features of PBR31, including the presence of a protein similar to the small subunit of D-family DNA polymerase and advanced lysis machinery. Taxonomic analysis showed the possibility of assigning phage PBR31 to a new taxon, although the complete taxonomic description of Xanthomonas phage PBR31 and other related bacteriophages is complicated by the complex evolutionary history of the formation of its genome. The general biological features of the PBR31 phage were analysed for the first time. Due to its presumably temperate lifestyle, there is doubt as to whether the PBR31 phage is appropriate for phage control purposes. Bioinformatics analysis, however, revealed the presence of cell wall-degrading enzymes that can be utilised for the treatment of bacterial infections.

Elongation and Ligation-Mediated Differential Coding for Label-Free and Locus-Specific Analysis of 8-Oxo-7,8-dihydroguanine in DNA.

Oxidative DNA damage is closely associated with the occurrence of numerous human diseases and cancers. 8-Oxo-7,8-dihydroguanine (8-oxoG) is the most prevalent form of DNA damage, and it has become not only an oxidative stress biomarker but also a new epigenetic-like biomarker. However, few approaches are available for the locus-specific detection of 8-oxoG because of the low abundance of 8-oxoG damage in DNA and the limited sensitivity of existing assays. Herein, we demonstrate the elongation and ligation-mediated differential coding for label-free and locus-specific analysis of 8-oxoG in DNA. This assay is very simple without the involvement of any specific labeled probes, complicated steps, and large sample consumption. The utilization of Bsu DNA polymerase can specifically initiate a single-base extension reaction to incorporate dATP into the opposite position of 8-oxoG, endowing this assay with excellent selectivity. The introduction of cascade amplification reaction significantly enhances the sensitivity. The proposed method can monitor 8-oxoG with a limit of detection of 8.21 × 10-19 M (0.82 aM), and it can identify as low as 0.001% 8-oxoG damage from a complex mixture with excessive undamaged DNAs. This method can be further applied to measure 8-oxoG levels in the genomic DNA of human cells under diverse oxidative stress, holding prospect potential in the dynamic monitoring of critical 8-oxoG sites, early clinical diagnosis, and gene damage-related biomedical research.

Discovery of a potent inhibitor, D-132, targeting AsfvPolX, via protein-DNA complex-guided pharmacophore screening and in vitro molecular characterizations.

The heightened transmissibility and capacity of African swine fever virus (ASFV) induce fatal diseases in domestic pigs and wild boars, posing significant economic repercussions and global threats. Despite extensive research efforts, the development of potent vaccines or treatments for ASFV remains a persistent challenge. Recently, inhibiting the AsfvPolX, a key DNA repair enzyme, emerges as a feasible strategy to disrupt viral replication and control ASFV infections. In this study, a comprehensive approach involving pharmacophore-based inhibitor screening, coupled with biochemical and biophysical analyses, were implemented to identify, characterize, and validate potential inhibitors targeting AsfvPolX. The constructed pharmacophore model, Phar-PolX-S, demonstrated efficacy in identifying a potent inhibitor, D-132 (IC50 = 2.8 ± 0.2 µM), disrupting the formation of the AsfvPolX-DNA complex. Notably, D-132 exhibited strong binding to AsfvPolX (KD = 6.9 ± 2.2 µM) through a slow-on-fast-off binding mechanism. Employing molecular modeling, it was elucidated that D-132 predominantly binds in-between the palm and finger domains of AsfvPolX, with crucial residues (R42, N48, Q98, E100, F102, and F116) identified as hotspots for structure-based inhibitor optimization. Distinctively characterized by a 1,2,5,6-tetrathiocane with modifications at the 3 and 8 positions involving ethanesulfonates, D-132 holds considerable promise as a lead compound for the development of innovative agents to combat ASFV infections.