Pole Research Articles

First report of saffron-associated mastrevirus 1 from saffron in Iran.

In spring 2022, 40 leaf samples of saffron plants harboring a wide variety of symptoms, including curling, yellowing, mosaic, dwarfing and leaf malformation were collected from three Khorasan provinces in Iran. These samples were processed using the virion-associated nucleic acid-based metagenomics approach (Moubset et al., 2022). Noteworthy, 147 contigs with a size >500 bp distributed among 35 samples with multiple symptomatic patterns (curling, yellowing, mosaic, dwarfing and leaf malformation) shared >90% nucleotide identity with saffron-associated mastrevirus 1 (SaM1) (Martínez-Fajardo et al., 2024). This virus has recently been detected from transcriptomic datasets from saffron (Crocus sativus L.) collected in India. SaM1 was proposed to belong to a new species of the Mastrevirus genus (Geminiviridae family) and was tentatively named Saffron-associated mastrevirus 1 (Martínez-Fajardo et al., 2024). In addition, contigs assigned to saffron potyviruses, including saffron latent virus, turnip mosaic virus and bean yellow mosaic virus were assembled from the 40 saffron samples pinpointing that no conclusion could be made on the causal virus of observed symptoms. Total DNAs of the 40 saffron samples were further extracted using the DNeasy Plant Mini Kit (Qiagen) and were tested for the presence of SaM1 using a primer pair amplifying a 327 bp long fragment located in the coat protein of SaM1 (CSAV_2F 5'-TTTAAGTCAGGGTCTGGAGATG-3'and CSAV_3R 5'-GGCATACTGTAACCTCGTCTTC). Amplification conditions were: an initial denaturation at 95°C for 10 min, 30 cycles at 95°C for 30 sec, 55°C for 30 sec, 72°C for 45 sec, and a final extension step at 72°C for 10 min. This PCR test confirmed that 28/40 samples tested positive for SaM1. Total DNAs from one sample (#69-32) testing positive for SaM1 was further amplified using Phi29 DNA polymerase (TempliPhi, GE Healthcare) by rolling circle amplification (RCA) as previously described (Shepherd et al., 2008). RCA products were used as a template for PCR amplification of the complete genome of SaM1 using abutting primers (CSAV_PST1F 5'-CTGCAGTTGCGGTAAGTCTATGTTGGCTG-3' and CSAV_PST1R 5'- CTGCAGAGACAACGATTCCCAAAATTACTTTGTTCC-3'). Amplification conditions were: an initial denaturation at 95°C for 10 min, 30 cycles at 95°C for 30 sec, 55°C for 1 min, 72°C for 2 min and 30 sec, and a final extension step at 72°C for 10 min. Amplification products of approximately 2.7 Kbp were gel purified, ligated to pGEM-T Easy (Promega) and sequenced by standard Sanger sequencing using a primer walking approach (Azenta, Germany). The arrangement of open reading frames within the 2,724 nt circular DNA of the CSAV_A9PstI clone (accession number PQ392009) is similar to those reported for mastreviruses, including the coat protein (CP), the movement protein (MP), the replication-associated protein (Rep) and the RepA protein. In addition, CSAV_A9PstI sequence shares 95.3% genome-wide identity with SaM1 consensus sequence assembled from transcriptomics data (Martínez-Fajardo et al., 2024). Overall, these results indicate that SaM1 is widely present in saffron in different regions of Northeast Iran. In addition, while all tested plant were infected by several viruses, the etiology of SaM1 remains to be determined precisely. To our knowledge, this is the first report of saffron-associated mastrevirus 1 from saffron in Iran.

A Case Report : Recurrent Herpes Labialis In A 12 Year Old Boy

Introduction: Herpes labialis, commonly known as cold sores, is an infection predominantly caused by herpes simplex type 1 (HSV-1). Transmission of herpes labialis occurs through direct contact with active lesions or bodily fluids, including saliva, from infected individuals. Patients with herpes labialis often report prodromal symptoms before lesions appear, such as fever, fatigue, loss of appetite, and muscle aches. Localized symptoms, including pain, burning, or itching at the site of the impending eruption, are also common. This case report discusses a recurrent herpes labialis case at Wangaya Regional General Hospital, aiming to enhance understanding of the condition's presentation, management strategies to prevent recurrence, and measures to minimize transmission. Case Report : A 12-year-old boy was diagnosed with recurrent herpes labialis caused by an infection with herpes simplex virus type 1 (HSV-1). This diagnosis was based on the patient's medical history, which indicated prodromal symptoms and polymorphic lesions, as well as dermatological examination findings that showed herpetiform vesicles with crusting. The patient’s history of similar symptoms and the lesion’s localization to the vermillion border of the lips support the diagnosis of a recurrent infection. Herpes labialis is most commonly seen in children, particularly those under 19 years of age, which corresponds with the patient’s age. The primary risk factor is direct contact with an infected individual, which aligns with the patient's history of frequently sharing food and drinks with a friend who also exhibited similar symptoms. Additionally, the patient was known to share electronic cigarettes (vapes) with schoolmates. Recurrent herpes labialis can be triggered by various factors, including weakened immunity and UV exposure, both of which were present in this case. Conclusion: The main treatment provided for this patient was oral acyclovir. The use of acyclovir aims to reduce symptoms, accelerate healing, and lower the risk of infection transmission. Acyclovir works by inhibiting viral replication through the suppression of DNA polymerase, which reduces the amount of active virus and speeds up recovery. The dosage of acyclovir given to the patient was in accordance with recommended guidelines. Supportive diagnostic tests, such as serology or Tzanck smear, are recommended to confirm the diagnosis. However, in this case, the diagnosis was already established through medical history and physical examination. Given that this is a contagious disease, the patient was educated on measures to reduce the risk of transmission.

Guardians of the Genome: Iron–Sulfur Proteins in the Nucleus

Iron–sulfur (Fe-S) clusters are essential cofactors found in many proteins in the mitochondria, cytosol, and nucleus of the cell. These versatile cofactors may undergo reversible oxidation–reduction reactions to enable electron transfers; they may be structural and confer stability to a folded protein; they may be regulatory and transduce an iron signal that alters the function or stability of a recipient protein. Of the nearly 70 proteins described in mammalian cells that bind Fe-S clusters, about half localize exclusively or partially to the nucleus, where they are required for DNA replication and repair, telomere maintenance, transcription, mitosis, and cell cycle control. Most nuclear Fe-S cluster proteins interact with DNA, including DNA polymerases, primase, helicases, and glycosylases. However, the specific roles of the clusters in the enzymatic activities of these proteins and their interplay with DNA remain a matter of debate. Defects in the metallation of nuclear Fe-S proteins cause genome instability and alter the regulation of cell division and proliferation, which are hallmarks of various genetic diseases and cancers. Here, we provide an inventory of the nuclear Fe-S cluster-binding proteins and discuss cluster types, binding sites, the process of cluster acquisition, and the potential roles of the cluster in the function of the proteins. However, many questions remain unresolved. We highlight critical gaps in our understanding of cluster delivery to nuclear client proteins, the potential for cluster repair, and the mechanistic roles that clusters play in these enzymes. Taken together, this review brings the focus to the nucleus of the human cell as a hotspot for Fe-S cluster proteins and aims to inspire new research on the roles of iron in DNA metabolism and the maintenance of genome integrity.

Exquisite selectivity of griselimycin extends to beta subunit of DNA polymerases from Gram-negative bacterial pathogens

Griselimycin, a cyclic depsidecapeptide produced by Streptomyces griseus, is a promising lead inhibitor of the sliding clamp component of bacterial DNA polymerases (β-subunit of Escherichia coli DNA pol III). It was previously shown to inhibit the Mycobacterium tuberculosis β-clamp with remarkably high affinity and selectivity – the peptide lacks any interaction with the human sliding clamp. Here, we used a structural genomics approach to address the prospect of broader-spectrum inhibition, in particular of β-clamps from Gram-negative bacterial targets. Fifteen crystal structures of β-clamp orthologs were solved, most from Gram-negative bacteria, including eight cocrystal structures with griselimycin. The ensemble of structures samples widely diverse β-clamp architectures and reveals unique protein-ligand interactions with varying degrees of complementarity. Although griselimycin clearly co-evolved with Gram-positive β-clamps, binding affinity measurements demonstrate that the high selectivity observed previously extends to the Gram-negative orthologs, with KD values ranging from 7 to 496 nM for the wild-type orthologs considered. The collective results should aid future structure-guided development of peptide antibiotics against β-clamp proteins of a wide variety of bacterial targets.

Necrosis drives susceptibility to Mycobacterium tuberculosis in Polg D257A mutator mice.

The genetic and molecular determinants that underlie the heterogeneity of Mycobacterium tuberculosis (Mtb) infection outcomes in humans are poorly understood. Multiple lines of evidence demonstrate that mitochondrial dysfunction can exacerbate mycobacterial disease severity and mutations in some mitochondrial genes confer susceptibility to mycobacterial infection in humans. Here, we report that mutations in mitochondria DNA (mtDNA) polymerase gamma (POLG) potentiate susceptibility to Mtb infection in mice. Polg D257A mutator mtDNA mice fail to mount a protective innate immune response at an early infection timepoint, evidenced by high bacterial burdens, reduced M1 macrophages, and excessive neutrophil infiltration in the lungs. Immunohistochemistry reveals signs of enhanced necrosis in the lungs of Mtb-infected Polg D257A mice and Polg D257A mutator macrophages are hyper-susceptible to extrinsic triggers of necroptosis ex vivo . By assigning a role for mtDNA mutations in driving necrosis during Mtb infection, this work further highlights the requirement for mitochondrial homeostasis in mounting balanced immune responses to Mtb.

Integrated Analysis of Polymerase Family Gene Mutations in Acute Myeloid Leukemia: Clinical Features, Prognosis, and Bioinformatics Insights

Background and Objectives: The long-term prognosis of acute myeloid leukemia (AML) is challenging due to limited understanding of the molecular markers involved in its development. This study investigates the role of DNA polymerases in AML to offer new insights for diagnosis and treatment. Materials and Methods: A retrospective study on pediatric AML patients with POL gene family mutations from 2021 to 2024 was conducted. Patients were categorized based on risk stratification criteria, and the DAH regimen was used for induction chemotherapy. Bioinformatics analysis integrated data from various databases to identify key genes and develop survival analysis plots and AUC curves. Results: The study included 59 pediatric AML patients, revealing no significant differences in demographic or clinical characteristics between those with and without POL family gene mutations. However, patients with POL gene mutations showed higher complete remission rates after initial DAH chemotherapy (91.67% vs. 59.57%, p = 0.03607), indicating a potential treatment benefit. High expression of four POL genes (POLD1, POLE, POLG, and POLQ) in bone marrow and immune cells suggests their crucial role in hematopoiesis and immune response. Survival analysis across different datasets indicated that AML patients with overexpressed POL family genes had significantly worse outcomes, proposing these genes as potential prognostic biomarkers for AML. Conclusions: This study on pediatric AML demonstrates that POL gene family mutations are associated with higher remission rates post-chemotherapy, indicating their potential as prognostic markers. Bioinformatics analysis emphasizes the significance of these mutations in AML, highlighting their impact on disease prognosis.

First molecular report of the Chelonid alphaherpesvirus 5 (ChHV5 Scutavirus chelonidalpha5) in a green turtle (Chelonia mydas) with fibropapillomatosis in the southwest Gulf of Mexico

Fibropapillomatosis (FP) is an emerging neoplastic disease associated with chelonid herpesvirus 5 (ChHV5; Scutavirus chelonidalpha 5) that affects all species of marine turtles worldwide, mainly green turtles (Chelonia mydas) at coastal feeding sites. This report describes the case of a juvenile green turtle stranded alive on the coast of Veracruz, Mexico that presented 41 lesions suggestive of FP distributed on the eyes, neck, front flippers, axillary/inguinal regions and plastron. Morphologically, the lesions varied in size, shape and appearance of the surface. A tumour was collected and analysed by histopathology revealing a benign neoplasm with fibropapilloma characteristics (dermal and epidermal proliferation) and cytopathic effects consistent with herpesvirus infection, such as ballooning, reticular, and vacuolar degeneration, cell necrosis, eosinophilic intranuclear inclusion bodies, and inflammatory cell infiltration. The tumour tested positive for ChHV5 through conventional PCR targeting the UL30, UL18, UL22, and UL27 genes. Phylogenetic analysis of the DNA Polymerase (UL30) placed the Veracruz variant in the Western Atlantic/Eastern Caribbean cluster along with sequences from Florida, Colombia, Barbados, and Brazil. Additional identification of the CMA1.1 DNA mitochondrial haplotype for this individual supports the connectivity between green turtles from the northern and southern regions of the Gulf of Mexico (GoM) and the Caribbean. It also suggests a potential risk route for ChHV5 infection. This report details the first case of FP linked to ChHV5 in Veracruz and the southwestern GoM. Further research on FP and ChHV5 in these areas is crucial due to their role as habitats for five sea turtle species across various life stages.

Application of semi-quantitative loop-mediated isothermal amplification for gene expression study in Expi293 cells.

BACKGROUND: Mammalian cell cultures play a key role in the pharmaceutical industry, requiring constant monitoring of the cell conditions during fermentation. In addition to monitoring of the physical-chemical parameters, it is important to evaluate the internal state of cells, for which gene expression analysis is used. Currently, quantitative reverse transcription polymerase chain reaction (qPCR) is the dominant method. However, the loop-mediated isothermal amplification (LAMP) technique also attracts attention due to its high specificity, sensitivity and reaction rate. LAMP is becoming a promising tool for rapid analysis of gene expression, especially under the conditions of limited biological material or a large volume of samples. AIM: Development of a technique for quantifying the expression level of target genes in human cell culture Expi293. METHODS: For LAMP, a recombinant large fragment of Bacillus stearothermophilus DNA polymerase (Bst-pol) was obtained, purified, and optimal reaction conditions were determined. SYBR Green I and LUCS13 were used as intercalating dyes. The amplification parameters for different concentrations of the enzyme and the dye were analyzed. Standard SYBR Green I kits were used for qPCR. Both methods were compared when analyzing the expression of IGF 1, FGF2 and EIF3i genes in cell lines with an increased expression level of these genes. RESULTS: It has been shown that using LUCS13 dye provides the classic S-shape of the signal accumulation curve in LAMP, while using SYBR Green I dye causes artifacts. The optimal concentration of Bst-pol was 40 ng/µl. When comparing the two methods, it was found that LAMP has greater sensitivity, allows determining gene expression with an accuracy comparable to qPCR, demonstrating a shorter reaction time (up to 35 minutes). CONCLUSION: Although quantitative PCR remains the main method for assessing the level of gene expression, loop-mediated isothermal amplification offers a number of advantages that make it an attractive alternative for various biotechnological purposes. Due to its high speed, ease of execution and accessibility, as well as high sensitivity and specificity, LAMP is a valuable technique for rapid analysis of gene expression during cell culture monitoring.

A Comprehensive Review of Pfu DNA Polymerase: Extraction, Production and Applications

Pfu DNA Polymerase, a thermostable enzyme derived from the hyperthermophilic archaeon Pyrococcus furiosus, is widely recognized for its high fidelity and strong processivity. Its 3'-5' exonuclease activity makes it indispensable for the correct amplification of both short and complex DNA strands. These biochemical properties of Pfu DNA Polymerase have encouraged considerable advancements in its extraction and production methods. This review covers some of the traditional approaches for purification, including protein purification and affinity chromatography, and updates on recent advances in recombinant gene expression, automated systems of production, and membrane-based technologies. New ways of engineering enzymes have recently been developed, such as CRISPR-Cas9-mediated gene optimization, which raised the bar on extraction efficiency to meet emerging demand. Once challenging, Pfu DNA Polymerase production has been significantly streamlined through recombinant expression in E. coli both at a laboratory and commercial scale. The optimization techniques that are involved with IPTG concentration and Response Surface Methodology have increased the yield by up to 30%. Auto-induction means even higher biomass outputs are allowed. Today, applications of Pfu DNA Polymerase span from standard PCR up to advanced clinical diagnostics in the fields of molecular biology, forensic analysis, clinical microbiology, and biotechnology.

TTF2 promotes replisome eviction from stalled forks in mitosis.

When cells enter mitosis with under-replicated DNA, sister chromosome segregation is compromised, which can lead to massive genome instability. The replisome-associated E3 ubiquitin ligase TRAIP mitigates this threat by ubiquitylating the CMG helicase in mitosis, leading to disassembly of stalled replisomes, fork cleavage, and restoration of chromosome structure by alternative end-joining. Here, we show that replisome disassembly requires TRAIP phosphorylation by the mitotic Cyclin B-CDK1 kinase, as well as TTF2, a SWI/SNF ATPase previously implicated in the eviction of RNA polymerase from mitotic chromosomes. We find that TTF2 tethers TRAIP to replisomes using an N-terminal Zinc finger that binds to phosphorylated TRAIP and an adjacent TTF2 peptide that contacts the CMG-associated leading strand DNA polymerase ε. This TRAIP-TTF2-pol ε bridge, which forms independently of the TTF2 ATPase domain, is essential to promote CMG unloading and stalled fork breakage. Conversely, RNAPII eviction from mitotic chromosomes requires the ATPase activity of TTF2. We conclude that in mitosis, replisomes undergo a CDK- and TTF2-dependent structural reorganization that underlies the cellular response to incompletely replicated DNA.

Identification of biallelic POLA2 variants in two families with an autosomal recessive telomere biology disorder

AbstractPOLA2 encodes the accessory subunit of DNA polymerase α (polα)/primase, which is crucial for telomere C-strand fill-in. Incomplete fill-in of the C-rich telomeric strand after DNA replication has been proposed as a mechanism for Coats plus syndrome, a phenotype within the broader spectrum of telomere biology disorders (TBD). Coats plus syndrome has so far been associated with pathogenic variants in POT1, CTC1, and STN1. Here we report the findings of biallelic deleterious rare variants in POLA2 gene detected by whole genome sequencing and segregation analysis in five young adults from two unrelated families. All five individuals displayed abnormally short telomeres and a clinical phenotype suggesting a TBD disorder with Coats plus features including retinal and gastrointestinal telangiectasias. Our results suggest POLA2 as a novel autosomal recessive gene for a TBD with Coats plus features.

Emergence and persistence of Cyvirus cyprinidallo 2 (CyHV-2) in Prussian carp in Serbian lakes

Between 2018 and 2021, significant die-offs of Prussian carp (Carassius gibelio) occurred in five lakes in eastern, central and northern Serbia. In all cases, Cyvirus cyprinidallo 2, formerly named Cyprinid herpesvirus 2 (CyHV-2) infection was suspected, based on initial diagnostic observations. To determine the cause of these mass mortalities, real-time PCR assays targeting CyHV-2, CyHV-3, and SVCV were conducted. CyHV-2 DNA was detected in the affected populations, while SVCV and CyHV-3 were negative in the assay. The CyHV-2 was also detected in asymptomatic Prussian carp. Sequence analysis of the CyHV-2 DNA polymerase and helicase genes revealed high similarity to previously reported strains from Serbia and other countries. The occurrence of these outbreaks in Prussian carp populations in lakes indicates the probable spread of the virus within freshwater ecosystems in Serbia, potentially impacting the degradation of aquatic habitats.

Multiple Displacement Amplification Facilitates SMRT Sequencing of Microscopic Animals and the Genome of the Gastrotrich Lepidodermella squamata (Dujardin, 1841).

Obtaining adequate DNA for long-read genome sequencing remains a roadblock to producing contiguous genomes from small-bodied organisms, hindering understanding of phylogenetic relationships and genome evolution. Multiple displacement amplification (MDA) leverages Phi29 DNA polymerase to produce micrograms of DNA from picograms of input. MDA's inherent biases in amplification related to GC and repeat content and chimera production are a problem for long-read genome assembly, which has been little investigated. We explored the utility of MDA for generating template DNA for HiFi sequencing directly from living cells of Caenorhabditis elegans (Nematoda) and Lepidodermella squamata (Gastrotricha) containing one order of magnitude less DNA than required for the PacBio Ultra-Low DNA Input Workflow. HiFi sequencing of libraries prepared from MDA DNA resulted in highly contiguous and complete genomes for both C. elegans (102 Mbp assembly; 336 contigs; N50=868 Kbp; L50=39; BUSCO_nematoda_nucleotide: S:96.1%, D:2.8%) and L. squamata (122 Mbp assembly; 157 contigs; N50=3.9 Mbp; L50=13; BUSCO_metazoa_nucleotide: S:80.8%, D:2.8%). Coverage uniformity for reads from MDA DNA (Gini Index: 0.14, normalized mean across all 100 Kbp blocks: 0.49) and reads from pooled nematode DNA (Gini Index: 0.16, normalized mean across all 100 Kbp blocks: 0.49) proved similar. Using this approach, we sequenced the genome of the microscopic invertebrate Lepidodermella squamata (Gastrotricha), the first of its phylum. Using the newly sequenced genome, we infer Gastrotricha's long-debated phylogenetic position as the sister taxon of Platyhelminthes and conduct a comparative analysis of the Hox cluster.

Characterization and PCR Application of Family B DNA Polymerases from Thermococcus stetteri

DNA polymerases from the hyperthermophilic Archaea have attracted considerable attention as PCR enzymes due to their high thermal stability and proofreading 3′ → 5′ exonuclease activity. This study is the first to report data concerning the purification and biochemical characteristics of the Tst DNA polymerase from Thermococcus stetteri. Both the wild type Tst(wt) DNA polymerase and its chimeric form containing the P36H substitution—which reduces the enzyme’s affinity for the U-containing template and dUTP—and the DNA-binding domain Sso7d from S. solfataricus were obtained and analyzed. It was shown that Tst(wt) could effectively amplify up to 6-kb DNA fragments, whereas TstP36H–Sso7d could amplify DNA fragments up to 15 kb. It was found that TstP36H–Sso7d has superior PCR efficiency compared to the commonly used DNA polymerase PfuV93Q–Sso7d. For the amplification of a 2-kb DNA fragment, TstP36H–Sso7d required less than 10 s of extension time, whereas for PfuV93Q–Sso7d, the extension time was no less than 30 s. Steady-state kinetic assays revealed that the dNTP-binding affinity KdNTPm was the same for TstP36H–Sso7d and PfuV93Q–Sso7d, whereas the maximum rate of dNTP incorporation, kcat, was two orders of magnitude higher for TstP36H–Sso7d. Moreover, the incorporation of incorrect dNTP was not observed for TstP36H–Sso7d up to 56 °C, whereas for PfuV93Q–Sso7d, the extension of primer with incorrect dNTP was observed at 37 °C, supporting higher fidelity of TstP36H–Sso7d. The obtained data suggest that TstP36H–Sso7d may be a good candidate for high-fidelity DNA amplification.

Expression of Viral DNA Polymerase in Synthetic Recombinant Adeno-Associated Virus Producer Cell Line Enhances Full Particle Productivity.

Recombinant adeno-associated virus (rAAV) is a widely used viral vector in gene therapy. To meet the growing clinical demand, a scalable production technology which can efficiently produce high-quality products is required. We have developed a synthetic biology strategy to generate HEK293-based cell lines which have integrated essential AAV and adenoviral helper genes and are capable of producing rAAV upon induction. One such cell line, GX6B, produced up to 106 capsids per cell, but only a much lower level of rAAV genomes. The low AAV genome titer limited its rAAV productivity and increased empty viral particle content. To boost AAV genome amplification, the coding sequence of the DNA polymerase complex (UL30/UL42) from helper Herpes Simplex Virus type 1 (HSV-1) was placed under an inducible promoter control and integrated into GX6B genome at a relatively low level. The resulting clones produced significantly higher titer of viral genomes, while their capsid level was unaffected. As a result, the encapsidated rAAV2 titer and the full particle content were significantly increased. We further demonstrated that this strategy of expressing HSV-1 DNA polymerase to increase full particle productivity could be implemented in a synthetic cell line producing another serotype rAAV8.

Unveiling the Mechanism of Deprotonation and Proton Transfer of DNA Polymerase Catalysis via Single-Molecule Conductance.

DNA polymerases (Pols) play important roles in the transmission of genetic information. Although the function and (de)regulation of Pols are linked to many human diseases, the key mechanism of 3'-OH deprotonation and the PPi formation are not totally clear. In this work, a method is presented to detect the full catalytic cycle of human Pol (hPol β) in graphene-molecule-graphene single-molecule junctions. Real-time in situ monitoring successfully revealed the spatial and temporal properties of the open and closed conformation states of hPol β, distinguishing the reaction states in the Pols catalytic cycle and unveiling 3'-OH deprotonation and pyrophosphate (PPi) formation mechanism of hPol β. Proton inventory experiment demonstrated that the rate-limiting step of PPi formation is deprotonation, which occurs before a reverse conformational change. Additionally, by detecting the acidity (pKa), it is found that MgA-bound OH- acted as a general base and activated the nucleophile of 3'-OH, and that acidic residue D190 or D192 coordinated with MgB as a proton donor to PPi. This work provides useful insights into a fundamental chemical reaction that impacts genome synthesis efficiency and Pol fidelity, which the discovery of Pol-targeting drugs and design of artificial Pols for DNA synthetic applications are expected to accelerated.

Expression and functional study of DNA polymerases from Psychrobacillus sp. BL-248-WT-3 and FJAT-21963.

The properties of DNA polymerases isolated from thermophilic and mesophilic microorganisms, such as the thermophilic Geobacillus stearothermophilus (Bst) and mesophilic Bacillus subtilis phage (Phi29), have been widely researched. However, DNA polymerases in psychrophilic microorganisms remain poorly understood. In this study, we present for the first time the expression and functional characterization of DNA polymerases PWT-WT and FWT-WT from Psychrobacillus sp. BL-248-WT-3 and FJAT-21963. Enzymatic activity assays revealed that FWT-WT possessed strand displacement but lacked exonuclease activity and high ionic strength tolerance, whereas PWT-WT lacked all these properties. Further protein engineering and biochemical analysis identified D423 and S490 as critical mutation sites for improving strand displacement and tolerance to high ionic strength, specifically in the presence of 0-0.3 M potassium chloride (KCl), sodium chloride (NaCl), and potassium acetate (KAc). Three-dimensional structural analysis demonstrated that the size and the electric charge of the single-stranded DNA (ssDNA) encapsulation entrance were pivotal factors in the binding of the ssDNA template.

POLD3 as Controller of Replicative DNA Repair.

Multiple modes of DNA repair need DNA synthesis by DNA polymerase enzymes. The eukaryotic B-family DNA polymerase complexes delta (Polδ) and zeta (Polζ) help to repair DNA strand breaks when primed by homologous recombination or single-strand DNA annealing. DNA synthesis by Polδ and Polζ is mutagenic, but is needed for the survival of cells in the presence of DNA strand breaks. The POLD3 subunit of Polδ and Polζ is at the heart of DNA repair by recombination, by modulating polymerase functions and interacting with other DNA repair proteins. We provide the background to POLD3 discovery, investigate its structure, as well as function in cells. We highlight unexplored structural aspects of POLD3 and new biochemical data that will help to understand the pivotal role of POLD3 in DNA repair and mutagenesis in eukaryotes, and its impact on human health.

Genetic Characterization of a Novel Retron Element Isolated from Vibrio mimicus.

Bacterial reverse transcriptase coding gene (RT) is essential for the production of a small satellite DNA-RNA complex called multicopy single-stranded DNA (msDNA). In this study, we found a novel retron, retron-Vmi1 (Vm85) from Vibrio mimicus. The retron is comprised of the msr-msd region, orf323, and the ret gene, a genetic organization similar to Salmonella's retron-Sen2 (St85). The protein sequence of the RNA-directed DNA polymerase (RT-Vmi1) is highly homologous to the RTs of Vibrio metoecus, Vibrio parahaemolyticus, and Vibrio vulnificus. Phylogenetic and protein sequence similarity analysis of retron-Vmi1 ORF323 and RT revealed a close relatedness to retron-Sen2. We found that retron-Vmi1 was inserted in the dusA gene, similar to the insertion of the retron-Vpa1 (Vp96) of V. parahaemolyticus AQ3354, suggesting that retrons can be transferred via the tRNA gene. These results are the first convincing evidence that retron is moving across species. The neighboring genes of retron-Vmi1 shared high homology with the genetic environment of V. parahaemolyticus and V. vulnificus retrons. We also found two junction points within the retron-Vmi1 and the dusA gene suggesting that retron-Vmi1 was inserted into this site in a two-step manner.

Development of a chemiluminescent detection method for absolute activity measurement of Taq DNA polymerase through dATP consumption

Being one of the most extensively researched and widely used thermostable DNA polymerases, Taq DNA polymerase derived from Thermus aquaticus has played pivotal roles in numerous molecular biology applications. The accurate and convenient determination of enzymatic activity is a fundamental prerequisite for both academic research and the industrial application of Taq DNA polymerase. To address the limitations of traditional methods for polymerase activity determination, such as low accuracy, complicated operation, and radioactive hazards, we have devised an absolute-quantitation method for Taq DNA polymerase based on chemiluminescence detection of the consumption of dATP (CDC-dATP). The CDC-dATP method offers several advantages, including simplicity suitable for routine laboratory use, high sensitivity (detecting as low as 0.0025 U/μL), excellent reproducibility, a broad linear range for Taq DNA polymerase (0.0025–0.1 U/μL) with a strong linear relationship (R2 > 0.99), and, significantly, absolute activity quantitation without the associated radioactive hazards. We delved into the molecular behavior of Taq DNA polymerase in the CDC-dATP method. Furthermore, we explored the extensive application potential of the CDC-dATP methodology for measuring the activity of various other enzymes involved in dATP/ATP generation or consumption, encompassing other DNA polymerases, reverse transcriptases, RNA polymerases, hydrolases, and protein kinases, covering all seven major categories of enzymes.