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At least 19 recordsLinked to original sources
PubMed2025

An orthogonal T7 replisome for continuous hypermutation and accelerated evolution in E. coli.

Systems that perform continuous hypermutation of designated genes without compromising the integrity of the host genome can substantially accelerate the evolution of new or enhanced protein functions. We describe an orthogonal DNA replication system in Escherichia coli based on the controlled expression of the replisome of bacteriophage T7 (T7-ORACLE). The system replicates circular plasmids that enable high transformation efficiencies and seamless integration into standard molecular biology workflows. Engineering of T7 DNA polymerase yielded variant proteins with mutation rates of 1.7 × 10-5 substitutions per base in vivo-100,000-fold above the genomic mutation rate. We demonstrated continuous evolution using the T7 replisome by expanding the substrate scope of TEM-1 β-lactamase and increasing activity 5000-fold against clinically relevant monobactam and cephalosporin antibiotics in less than 1 week.

Bacteriophage T7↗
PubMed2025

Mapping Active RNA Polymerases in Proliferating and Quiescent Fission Yeast Cells Using Precision Run-On Sequencing.

The development of next-generation sequencing (NGS) approaches to investigate the functioning of RNA polymerases has led to groundbreaking advances in the field of transcriptional regulation. One powerful method, Precision nuclear Run-On sequencing (PRO-seq), maps the locations of RNA polymerase active sites genome-wide at high resolution. PRO-seq provides a snapshot of strand-specific transcriptional activity and does not rely on immunoprecipitation of the polymerase of interest. Notably, this technique has been utilized to investigate the control of the RNA polymerase II transcription cycle in a variety of model systems. However, the initially published PRO-seq method required significant amounts of starting sample and was technically challenging, both of which were deterrents for its broader use. Recently, an improved and simplified version called qPRO-seq that reduced the length of the experiment and the quantity of necessary input sample was developed for human and Drosophila cell lines. Here we provide an updated, step-by-step protocol in which we have validated and optimized qPRO-seq for the fission yeast Schizosaccharomyces pombe. Importantly, we have implemented this method for assessing RNA polymerase activity in nutrient-limiting conditions, for both proliferating and nitrogen-depleted quiescent cells.

Schizosaccharomyces↗
PubMed2025

Establishment of reverse genetics systems for Colorado tick fever virus.

The Colorado tick fever virus (CTFV), which has 12-segmented double-stranded RNA genomes, is a pathogenic arbovirus that causes severe diseases in humans. However, little progress has been made in the analysis of replication mechanisms and pathogenicity. This virological constraint is due to the absence of a reverse genetics system for CTFV; therefore, we aimed to establish the system. Initially, the efficacy of CTFV replication was investigated in various cell lines. CTFV was found to grow in many cell types derived from different hosts and organs. Subsequently, BHK-T7 cells stably expressing T7 RNA polymerase were transfected with plasmids encoding each of the 12 CTFV gene segments, expression plasmids encoding all CTFV proteins, and a vaccinia virus RNA-capping enzyme. Following transfection, the cells were co-cultured with Vero or HeLa cells. Using this system, we rescued monoreassortants and recombinant viruses harboring peptide-tagged viral proteins. Furthermore, an improved system using Expi293F cells expressing T7 RNA polymerase was established, which enabled the generation of recombinant reporter CTFVs. In conclusion, these reverse genetics systems for CTFV will greatly contribute to the understanding of viral replication mechanisms, pathogenesis, and transmission, ultimately facilitating the development of rational treatments and candidate vaccines.

Animals↗
PubMed2024

NS2 induces an influenza A RNA polymerase hexamer and acts as a transcription to replication switch.

Genome transcription and replication of influenza A virus (FluA), catalyzed by viral RNA polymerase (FluAPol), are delicately controlled across the virus life cycle. A switch from transcription to replication occurring at later stage of an infection is critical for progeny virion production and viral non-structural protein NS2 has been implicated in regulating the switch. However, the underlying regulatory mechanisms and the structure of NS2 remained elusive for years. Here, we determine the cryo-EM structure of the FluAPol-NS2 complex at ~3.0 Å resolution. Surprisingly, three domain-swapped NS2 dimers arrange three symmetrical FluPol dimers into a highly ordered barrel-like hexamer. Further structural and functional analyses demonstrate that NS2 binding not only hampers the interaction between FluAPol and the Pol II CTD because of steric conflicts, but also impairs FluAPol transcriptase activity by stalling it in the replicase conformation. Moreover, this is the first visualization of the full-length NS2 structure. Our findings uncover key molecular mechanisms of the FluA transcription-replication switch and have implications for the development of antivirals.

Viral Nonstructural Proteins↗
PubMed2026

Molecular Characterisation of Treacher Collins Syndrome in a South African Cohort: Novel Disease-Causing Variants in TCOF1 and POLR1D.

BACKGROUND: Treacher Collins syndrome (TCS) is a rare craniofacial disorder characterised by variable expressivity. It is caused by pathogenic variants in the TCOF1, POLR1D, POLR1C, or POLR1B genes. Common clinical features include hypoplasia of the zygomatic complex and mandible, downward-slanting palpebral fissures, lower eyelid anomalies, microtia, and hearing loss. Owing to its phenotypic overlap with other craniofacial syndromes, molecular testing is essential for establishing an accurate diagnosis and guiding effective clinical management. METHODS: Ten South African patients with a suspected clinical diagnosis of TCS underwent targeted next-generation sequencing (NGS) using a custom gene panel including TCOF1, POLR1C, and POLR1D genes. Variants were classified according to ACMG/AMP guidelines, with validation by Sanger sequencing where necessary. RESULTS: Disease-causing variants were identified in six of the ten patients (60%). These included five heterozygous variants in TCOF1 and one homozygous variant in POLR1D. Notably, five of the six variants were identified for the first time in this study. Additionally, a recurrent TCOF1 deletion was identified for the first time in an African family. CONCLUSION: This study expands the mutational spectrum of TCS in general and provides African data in particular. Findings support the use of panel-based NGS for diagnosis in resource-limited settings and highlight the need for population-specific variant data to improve diagnostic accuracy, guide clinical care, and support genetic counselling for affected individuals and their families.

Humans↗
PubMed2024

Sen1: The Varied Virtues of a Multifaceted Helicase.

Several machineries concurrently work on the DNA, but among them RNA Polymerases (RNAPs) are the most widespread and active users. The homeostasis of such a busy genomic environment relies on the existence of mechanisms that allow limiting transcription to a functional level, both in terms of extent and rate. Sen1 is a central player in this sense: using its translocase activity this protein has evolved the specific function of dislodging RNAPs from the DNA template, thus ending the transcription cycle. Over the years, studies have shown that Sen1 uses this same mechanism in a multitude of situations, allowing termination of all three eukaryotic RNAPs in different contexts. In virtue of its helicase activity, Sen1 has also been proposed to have a prominent function in the resolution of co-transcriptional genotoxic R-loops, which can cause the stalling of replication forks. In this review, we provide a synopsis of past and recent findings on the functions of Sen1 in yeast and of its human homologue Senataxin (SETX).

Humans↗
PubMed2024

NusG-Spt5 Transcription Factors: Universal, Dynamic Modulators of Gene Expression.

The accurate and efficient biogenesis of RNA by cellular RNA polymerase (RNAP) requires accessory factors that regulate the initiation, elongation, and termination of transcription. Of the many discovered to date, the elongation regulator NusG-Spt5 is the only universally conserved transcription factor. With orthologs and paralogs found in all three domains of life, this ubiquity underscores their ancient and essential regulatory functions. NusG-Spt5 proteins evolved to maintain a similar binding interface to RNAP through contacts of the NusG N-terminal domain (NGN) that bridge the main DNA-binding cleft. We propose that varying strength of these contacts, modulated by tethering interactions, either decrease transcriptional pausing by smoothing the rugged thermodynamic landscape of transcript elongation or enhance pausing, depending on which conformation of RNAP is stabilized by NGN contacts. NusG-Spt5 contains one (in bacteria and archaea) or more (in eukaryotes) C-terminal domains that use a KOW fold to contact diverse targets, tether the NGN, and control RNA biogenesis. Recent work highlights these diverse functions in different organisms. Some bacteria contain multiple specialized NusG paralogs that regulate subsets of operons via sequence-specific targeting, controlling production of antibiotics, toxins, or capsule proteins. Despite their common origin, NusG orthologs can differ in their target selection, interacting partners, and effects on RNA synthesis. We describe the current understanding of NusG-Spt5 structure, interactions with RNAP and other regulators, and cellular functions including significant recent progress from genome-wide analyses, single-molecule visualization, and cryo-EM. The recent findings highlight the remarkable diversity of function among these structurally conserved proteins.

Archaea↗
PubMed2024

How do bacteria tune transcription termination efficiency?

Bacterial operons often contain intergenic transcription terminators that terminate some, but not all, RNA polymerase molecules. In these operons, the level of terminator readthrough determines downstream gene expression and helps establish protein ratios among co-regulated genes. Despite its critical role in maintaining stoichiometric gene expression, terminator strength remains difficult to predict from DNA sequence. The necessary features of a major class of bacterial terminators - intrinsic terminators - have been known for half a century, but a strong sequence-function model has yet to be developed. Here, we summarize high-throughput approaches for probing the sequence determinants of intrinsic termination efficiency and discuss the impact of trans-acting factors on this sequence-function relationship. Building on the main lessons from these studies, we map out the experimental challenges that must be circumvented to establish a quantitative model for termination efficiency.

Transcription Termination, Genetic↗
PubMed2026

Halorubrum marinum sp. nov., Halorubrum rarum sp. nov., Halorubrum wangae sp. nov., Halorubrum shenae sp. nov., and Halorubrum zhoui sp. nov., halophilic archaea from coastal tidal flats, a saline lake, and a marine solar saltern.

Five novel halophilic archaeal strains, designated DTA46T, DTA98T, HHNYT27T, N11T, and SY-15T, were isolated from diverse saline environments across various regions of China. Amplicon and metagenome analyses revealed that three amplicon reads were affiliated with strains DTA46T, HHNYT27T, and N11T while two MAGs related to strains N11T and SY-15T. The sequence similarities among these five strains and current species of the genus Halorubrum were 93.1%-99.1% and 86.0%-95.9% judged by 16S rRNA and rpoB' genes, respectively. Phylogenomic and comparative genomic analyses revealed their close affiliation with Halorubrum. The average nucleotide identity (ANI), digital DNA-DNA hybridization (dDDH), and average amino acid identity (AAI) values between these strains and existing Halorubrum species ranged from 74.9%-93.6%, 22.3%-58.3%, and 68.3%-93.7%, respectively. All are below the recommended thresholds for species delineation, which supports their classification as novel taxa. The growth characteristics of strains DTA46T, DTA98T, HHNYT27T, N11T, and SY-15T were determined as follows: temperature range 20-60 °C (optima: 35, 37-42, 37, 35, and 42 °C), NaCl concentration 1.4-5.5 M (optima: 2.6, 3.1, 3.1, 3.1, and 5.1 M), and pH range 5.5-9.5 (optima: 8.0, 8.0, 7.0, 7.5, and 7.0). Based on the polyphasic characterization integrating phenotypic, chemotaxonomic, phylogenetic, and phylogenomic evidence, strains DTA46T, DTA98T, HHNYT27T, N11T, and SY-15T are proposed to represent five novel species of the genus Halorubrum, for which the names Halorubrum marinum sp. nov., Halorubrum rarum sp. nov., Halorubrum wangae sp. nov., Halorubrum shenae sp. nov., and Halorubrum zhoui sp. nov. are designated, respectively.

Phylogeny↗
PubMed2026

Cold-adapted RNA polymerase from Pseudomonas phage Njord improves synthesis of therapeutic mRNA.

An RNA polymerase identified in the genome of Pseudomonas phage Njord offers a promising tool for the synthesis of mRNA and other therapeutic nucleic acids. Originating from a marine microbial ecosystem, Njord RNAP transcribes RNA at high yield even under low temperature conditions. Key properties of the enzyme relevant to mRNA synthesis are presented including transcriptional fidelity, promoter specificity, incorporation of modified nucleotides, and the impurity profile of the RNA. Specific attention is given to the formation of contaminating double-stranded RNA (dsRNA) species. Analysis of transcription reactions shows that DNA-templated promoter-independent transcription is a major source of detectable dsRNA impurities and that Njord RNAP displays a minimal level of this activity. Consistent with the known inflammatory role of dsRNA in synthetic mRNA, transcriptomic analysis of cell culture and a live animal study demonstrates that mRNA synthesized with Njord RNAP elicits only a minimal immune response. This natural enzyme enables efficient mRNA synthesis at ambient temperature and produces transcripts essentially free of dsRNA, offering significant potential to streamline mRNA manufacturing processes.

DNA-Directed RNA Polymerases↗
PubMed2026

Genomic insights into low-level rifampicin resistance mediated by borderline rpoB mutations in Mycobacterium tuberculosis: prevalence and phylogeny in Northeast China.

The emergence of low-level rifampicin (RIF) resistance in Mycobacterium tuberculosis poses a challenge to tuberculosis (TB) control, as it often leads to discordance between genotypic resistance detected by molecular assays (e.g., Xpert MTB/RIF) and phenotypic susceptibility in conventional drug susceptibility testing (DST). In this study, we performed whole-genome sequencing (WGS) on 17 clinical isolates from Changchun, Northeast China, which exhibited such discordance. All isolates harbored functional borderline mutations in the rpoB RRDR region, predominantly Leu452Pro and Leu430Pro (29% each), followed by His445Asn (18%). RIF minimum inhibitory concentration (MIC) values ranged from ≤0.25 to 1.0 mg/L, confirming low-level resistance. Notably, 53% (9/17) of the isolates were co-resistant to fluoroquinolones and 24% (4/17) to isoniazid (INH). According to WHO classification, 59% (10/17) were pre-extensively drug-resistant TB (Pre-XDR-TB) or multidrug-resistant TB (MDR-TB). Phylogenetic analysis revealed that 94% (16/17) belonged to the East Asian Beijing lineage (Lineage 2.2.1), with no evidence of recent local transmission. These findings underscore the complexity of low-level RIF resistance and its frequent association with broader drug resistance in a dominant lineage, highlighting the need for integrating MIC and WGS into diagnostic algorithms to guide appropriate treatment and surveillance.IMPORTANCEThe accurate detection of RIF resistance is critical for the management of TB, yet standard phenotypic methods often fail to identify strains with low-level resistance conferred by borderline rpoB mutations. This study provides the first genomic characterization of such discordant isolates in Northeast China, revealing a high prevalence of co-resistance to other key drugs and a strong association with the locally dominant Beijing lineage. The findings emphasize that reliance on phenotypic DST alone may lead to underestimation of drug resistance and inappropriate treatment, potentially contributing to the emergence and spread of Pre-XDR-TB and MDR-TB. Incorporating MIC determination and WGS into routine diagnostics could enhance detection, inform tailored therapy, and improve surveillance of these clinically significant strains.

Mycobacterium tuberculosis↗
PubMed2026

Development and optimization of T-ARMS PCR assays for detection of lethal haplotypes of TADA2A, UR1B, and PORL1B in pigs in Vietnam.

Marker-assisted selection has increasingly relied on single-nucleotide polymorphisms (SNPs) as robust genetic markers, particularly in livestock breeding programs. In pig farming, embryonic mortality significantly affects litter size, and SNPs in reference genes have been implicated as potential causal factors. We developed and optimized a tetra-primer amplification refractory mutation system (T-ARMS) PCR assay for rapid, cost-effective detection of SNPs in 3 candidate genes-TADA2A, PORL1B, URB1-that are associated with embryonic lethality and reproductive performance. Primer sets were designed based on known mutation sites and validated using synthetic gene constructs and porcine genomic DNA from pigs of Duroc and Landrace breeds. Optimization of annealing temperatures and primer concentration ratios yielded distinct and reproducible allele-specific amplicon patterns that were corroborated by PCR-RFLP and Sanger sequencing. Our T-ARMS PCR protocol, which requires minimal equipment and reduces processing time to <3&#x2009;h, had high specificity and efficiency in differentiating wild-type, heterozygous, and homozygous mutant genotypes in 20 Duroc and 20 Landrace pigs. Our Tetra-ARMS PCR assay is a robust and economically viable tool for SNP genotyping in pig breeding programs, potentially contributing to the reduction of embryonic lethality and the improvement of overall reproductive outcomes.

Sus scrofa↗
PubMed2021

Mechanism, cellular functions and cancer roles of polymerase-theta-mediated DNA end joining.

Cellular pathways that repair chromosomal double-strand breaks (DSBs) have pivotal roles in cell growth, development and cancer. These DSB repair pathways have been the target of intensive investigation, but one pathway - alternative end joining (a-EJ) - has long resisted elucidation. In this Review, we highlight recent progress in our understanding of a-EJ, especially the assignment of DNA polymerase theta (Pol&#x3b8;) as the predominant mediator of a-EJ in most eukaryotes, and discuss a potential molecular mechanism by which Pol&#x3b8;-mediated end joining (TMEJ) occurs. We address possible cellular functions of TMEJ in resolving DSBs that are refractory to repair by non-homologous end joining (NHEJ), DSBs generated following replication fork collapse and DSBs present owing to stalling of repair by homologous recombination. We also discuss how these context-dependent cellular roles explain how TMEJ can both protect against and cause genome instability, and the emerging potential of Pol&#x3b8; as a therapeutic target in cancer.

Animals↗
PubMed2023

Pol&#x3b3; coordinates DNA synthesis and proofreading to ensure mitochondrial genome integrity.

Accurate replication of mitochondrial DNA (mtDNA) by DNA polymerase &#x3b3; (Pol&#x3b3;) is essential for maintaining cellular energy supplies, metabolism, and cell cycle control. To illustrate the structural mechanism for Pol&#x3b3; coordinating polymerase (pol) and exonuclease (exo) activities to ensure rapid and accurate DNA synthesis, we determined four cryo-EM structures of Pol&#x3b3; captured after accurate or erroneous incorporation to a resolution of 2.4-3.0&#x2009;&#xc5;. The structures show that Pol&#x3b3; employs a dual-checkpoint mechanism to sense nucleotide misincorporation and initiate proofreading. The transition from replication to error editing is accompanied by increased dynamics in both DNA and enzyme, in which the polymerase relaxes its processivity and the primer-template DNA unwinds, rotates, and backtracks to shuttle the mismatch-containing primer terminus 32&#x2009;&#xc5; to the exo site for editing. Our structural and functional studies also provide a foundation for analyses of Pol&#x3b3; mutation-induced human diseases and aging.

Humans↗
PubMed2024

N6-methyladenosine modification of a parvovirus-encoded small noncoding RNA facilitates viral DNA replication through recruiting Y-family DNA polymerases.

Human bocavirus 1 (HBoV1) is a human parvovirus that causes lower respiratory tract infections in young children. It contains a single-stranded (ss) DNA genome of ~5.5 kb that encodes a small noncoding RNA of 140 nucleotides known as bocavirus-encoded small RNA (BocaSR), in addition to viral proteins. Here, we determined the secondary structure of BocaSR in vivo by using DMS-MaPseq. Our findings reveal that BocaSR undergoes N6-methyladenosine (m6A) modification at multiple sites, which is critical for viral DNA replication in both dividing HEK293 cells and nondividing cells of the human airway epithelium. Mechanistically, we found that m6A-modified BocaSR serves as a mediator for recruiting Y-family DNA repair DNA polymerase (Pol) &#x3b7; and Pol &#x3ba; likely through a direct interaction between BocaSR and the viral DNA replication origin at the right terminus of the viral genome. Thus, this report represents direct involvement of a viral small noncoding RNA in viral DNA replication through m6A modification.

Humans↗
PubMed2024

DNA polymerase delta governs parental histone transfer to DNA replication lagging strand.

Chromatin replication is intricately intertwined with the recycling of parental histones to the newly duplicated DNA strands for faithful genetic and epigenetic inheritance. The transfer of parental histones occurs through two distinct pathways: leading strand deposition, mediated by the DNA polymerase &#x3b5; subunits Dpb3/Dpb4, and lagging strand deposition, facilitated by the MCM helicase subunit Mcm2. However, the mechanism of the facilitation of Mcm2 transferring parental histones to the lagging strand while moving along the leading strand remains unclear. Here, we show that the deletion of Pol32, a nonessential subunit of major lagging-strand DNA polymerase &#x3b4;, results in a predominant transfer of parental histone H3-H4 to the leading strand during replication. Biochemical analyses further demonstrate that Pol32 can bind histone H3-H4 both in vivo and in vitro. The interaction of Pol32 with parental histone H3-H4 is disrupted through the mutation of the histone H3-H4 binding domain within Mcm2. Our findings identify the DNA polymerase &#x3b4; subunit Pol32 as a critical histone chaperone downstream of Mcm2, mediating the transfer of parental histones to the lagging strand during DNA replication.

DNA Polymerase III↗
PubMed2024

Natural history of eukaryotic DNA viruses with double jelly-roll major capsid proteins.

The phylum Preplasmiviricota (kingdom Bamfordvirae, realm Varidnaviria) is a broad assemblage of diverse viruses with comparatively short double-stranded DNA genomes (<50 kbp) that produce icosahedral capsids built from double jelly-roll major capsid proteins. Preplasmiviricots infect hosts from all cellular domains, testifying to their ancient origin, and, in particular, are associated with six of the seven supergroups of eukaryotes. Preplasmiviricots comprise four major groups of viruses, namely, polintons, polinton-like viruses (PLVs), virophages, and adenovirids. We used protein structure modeling and analysis to show that protein-primed DNA polymerases (pPolBs) of polintons, virophages, and cytoplasmic linear plasmids encompass an N-terminal domain homologous to the terminal proteins (TPs) of prokaryotic PRD1-like tectivirids and eukaryotic adenovirids that are involved in protein-primed replication initiation, followed by a viral ovarian tumor-like cysteine deubiquitinylase (vOTU) domain. The vOTU domain is likely responsible for the cleavage of the TP from the large pPolB polypeptide and is inactivated in adenovirids, in which TP is a separate protein. Many PLVs and transpovirons encode a distinct derivative of polinton-like pPolB that retains the TP, vOTU, and pPolB polymerization palm domains but lacks the exonuclease domain and instead contains a superfamily 1 helicase domain. Analysis of the presence/absence and inactivation of the vOTU domains and replacement of pPolB with other DNA polymerases in eukaryotic preplasmiviricots enabled us to outline a complete scenario for their origin and evolution.

Capsid Proteins↗