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Nature Communications Aug 2023Bacteria colonize almost all parts of the human body and can differ significantly. However, the population level transcriptomics measurements can only describe the...
Bacteria colonize almost all parts of the human body and can differ significantly. However, the population level transcriptomics measurements can only describe the average bacteria population behaviors, ignoring the heterogeneity among bacteria. Here, we report a droplet-based high-throughput single-microbe RNA-seq assay (smRandom-seq), using random primers for in situ cDNA generation, droplets for single-microbe barcoding, and CRISPR-based rRNA depletion for mRNA enrichment. smRandom-seq showed a high species specificity (99%), a minor doublet rate (1.6%), a reduced rRNA percentage (32%), and a sensitive gene detection (a median of ~1000 genes per single E. coli). Furthermore, smRandom-seq successfully captured transcriptome changes of thousands of individual E. coli and discovered a few antibiotic resistant subpopulations displaying distinct gene expression patterns of SOS response and metabolic pathways in E. coli population upon antibiotic stress. smRandom-seq provides a high-throughput single-microbe transcriptome profiling tool that will facilitate future discoveries in microbial resistance, persistence, microbe-host interaction, and microbiome research.
Topics: Humans; Escherichia coli; High-Throughput Nucleotide Sequencing; RNA-Seq; Anti-Bacterial Agents; DNA Primers; RNA, Ribosomal
PubMed: 37612289
DOI: 10.1038/s41467-023-40137-9 -
Molecular Cell Aug 2023During eukaryotic DNA replication, Pol α-primase generates primers at replication origins to start leading-strand synthesis and every few hundred nucleotides during...
During eukaryotic DNA replication, Pol α-primase generates primers at replication origins to start leading-strand synthesis and every few hundred nucleotides during discontinuous lagging-strand replication. How Pol α-primase is targeted to replication forks to prime DNA synthesis is not fully understood. Here, by determining cryoelectron microscopy (cryo-EM) structures of budding yeast and human replisomes containing Pol α-primase, we reveal a conserved mechanism for the coordination of priming by the replisome. Pol α-primase binds directly to the leading edge of the CMG (CDC45-MCM-GINS) replicative helicase via a complex interaction network. The non-catalytic PRIM2/Pri2 subunit forms two interfaces with CMG that are critical for in vitro DNA replication and yeast cell growth. These interactions position the primase catalytic subunit PRIM1/Pri1 directly above the exit channel for lagging-strand template single-stranded DNA (ssDNA), revealing why priming occurs efficiently only on the lagging-strand template and elucidating a mechanism for Pol α-primase to overcome competition from RPA to initiate primer synthesis.
Topics: Humans; DNA Primase; Cryoelectron Microscopy; DNA Replication; DNA Helicases; Saccharomyces cerevisiae; DNA, Single-Stranded
PubMed: 37506699
DOI: 10.1016/j.molcel.2023.06.035 -
Nucleic Acids Research Aug 2023The discovery of reverse transcriptases (RTs) challenged the central dogma by establishing that genetic information can also flow from RNA to DNA. Although they act as...
The discovery of reverse transcriptases (RTs) challenged the central dogma by establishing that genetic information can also flow from RNA to DNA. Although they act as DNA polymerases, RTs are distantly related to replicases that also possess de novo primase activity. Here we identify that CRISPR associated RTs (CARTs) directly prime DNA synthesis on both RNA and DNA. We demonstrate that RT-dependent priming is utilized by some CRISPR-Cas complexes to synthesise new spacers and integrate these into CRISPR arrays. Expanding our analyses, we show that primer synthesis activity is conserved in representatives of other major RT classes, including group II intron RT, telomerase and retroviruses. Together, these findings establish a conserved innate ability of RTs to catalyse de novo DNA primer synthesis, independently of accessory domains or alternative priming mechanisms, which likely plays important roles in a wide variety of biological pathways.
Topics: DNA-Directed DNA Polymerase; HIV Reverse Transcriptase; Introns; Retroviridae; RNA; RNA-Directed DNA Polymerase; DNA Replication
PubMed: 37279911
DOI: 10.1093/nar/gkad478 -
Bioscience Reports Jul 2023To pass on genetic information to the next generation, cells must faithfully replicate their genomes to provide copies for each daughter cell. To synthesise these... (Review)
Review
To pass on genetic information to the next generation, cells must faithfully replicate their genomes to provide copies for each daughter cell. To synthesise these duplicates, cells employ specialised enzymes called DNA polymerases, which rapidly and accurately replicate nucleic acid polymers. However, most polymerases lack the ability to directly initiate DNA synthesis and required specialised replicases called primases to make short polynucleotide primers, from which they then extend. Replicative primases (eukaryotes and archaea) belong to a functionally diverse enzyme superfamily known as Primase-Polymerases (Prim-Pols), with orthologues present throughout all domains of life. Characterised by a conserved catalytic Prim-Pol domain, these enzymes have evolved various roles in DNA metabolism, including DNA replication, repair, and damage tolerance. Many of these biological roles are fundamentally underpinned by the ability of Prim-Pols to generate primers de novo. This review examines our current understanding of the catalytic mechanisms utilised by Prim-Pols to initiate primer synthesis.
Topics: DNA Primase; DNA-Directed DNA Polymerase; DNA Replication; Catalytic Domain; DNA
PubMed: 37358261
DOI: 10.1042/BSR20221986 -
Journal of Cachexia, Sarcopenia and... Dec 2023Exercise stimulates the activation of muscle satellite cells, which facilitate the maintenance of stem cells and their myogenic conversion during muscle regeneration....
BACKGROUND
Exercise stimulates the activation of muscle satellite cells, which facilitate the maintenance of stem cells and their myogenic conversion during muscle regeneration. However, the underlying mechanism is not yet fully understood. This study shows that the transcriptional co-activator with PDZ-binding motif (TAZ) stimulates muscle regeneration via satellite cell activation.
METHODS
Taz mice were crossed with the paired box gene 7 (Pax7) mice to generate muscle satellite cell-specific TAZ knockout (sKO) mice. Mice were trained in an endurance exercise programme for 4 weeks. Regenerated muscles were harvested and analysed by haematoxylin and eosin staining. Muscle tissues were also analysed by immunofluorescence staining, immunoblot analysis and quantitative reverse transcription PCR (qRT-PCR). For the in vitro study, muscle satellite cells from wild-type and sKO mice were isolated and analysed. Mitochondrial DNA was quantified by qRT-PCR using primers that amplify the cyclooxygenase-2 region of mitochondrial DNA. Quiescent and activated satellite cells were stained with MitoTracker Red CMXRos to analyse mitochondria. To study the p38 mitogen-activated protein kinase (MAPK)-TAZ signalling axis, p38 MAPK was activated by introducing the MAPK kinase 6 plasmid into satellite cells and also inhibited by treatment with the p38 MAPK inhibitor, SB203580.
RESULTS
TAZ interacts with Pax7 to induce Myf5 expression and stimulates mammalian target of rapamycin signalling for satellite cell activation. In sKO mice, TAZ depletion reduces muscle satellite cell number by 38% (0.29 ± 0.073 vs. 0.18 ± 0.034, P = 0.0082) and muscle regeneration. After muscle injury, TAZ levels (2.59-fold, P < 0.0001) increase in committed cells compared to self-renewing cells during asymmetric satellite cell division. Mechanistically, the polarity protein Pard3 induces TAZ (2.01-fold, P = 0.008) through p38 MAPK, demonstrating that the p38 MAPK-TAZ axis is important for muscle regeneration. Physiologically, endurance exercise training induces muscle satellite cell activation and increases muscle fibre diameter (1.33-fold, 43.21 ± 23.59 vs. 57.68 ± 23.26 μm, P = 0.0004) with increased TAZ levels (1.76-fold, P = 0.017). However, sKO mice had a 39% reduction in muscle satellite cell number (0.20 ± 0.03 vs. 0.12 ± 0.02, P = 0.0013) and 24% reduction in muscle fibre diameter compared to wild-type mice (61.07 ± 23.33 vs. 46.60 ± 24.29 μm, P = 0.0006).
CONCLUSIONS
Our results demonstrate a novel mechanism of TAZ-induced satellite cell activation after muscle injury and exercise, suggesting that activation of TAZ in satellite cells may ameliorate the muscle ageing phenotype and may be an important target protein for the drug development in sarcopenia.
Topics: Animals; Mice; DNA, Mitochondrial; Mammals; Muscle Fibers, Skeletal; p38 Mitogen-Activated Protein Kinases; Satellite Cells, Skeletal Muscle; Signal Transduction; Mitogen-Activated Protein Kinase 14
PubMed: 37923703
DOI: 10.1002/jcsm.13348 -
Molecular Ecology Dec 2023Exhaustive biodiversity data, covering all the taxa in an environment, would be fundamental to understand how global changes influence organisms living at different... (Review)
Review
Exhaustive biodiversity data, covering all the taxa in an environment, would be fundamental to understand how global changes influence organisms living at different trophic levels, and to evaluate impacts on interspecific interactions. Molecular approaches such as DNA metabarcoding are boosting our ability to perform biodiversity inventories. Nevertheless, even though a few studies have recently attempted exhaustive reconstructions of communities, holistic assessments remain rare. The majority of metabarcoding studies published in the last years used just one or two markers and analysed a limited number of taxonomic groups. Here, we provide an overview of emerging approaches that can allow all-taxa biological inventories. Exhaustive biodiversity assessments can be attempted by combining a large number of specific primers, by exploiting the power of universal primers, or by combining specific and universal primers to obtain good information on key taxa while limiting the overlooked biodiversity. Multiplexes of primers, shotgun sequencing and capture enrichment may provide a better coverage of biodiversity compared to standard metabarcoding, but still require major methodological advances. Here, we identify the strengths and limitations of different approaches, and suggest new development lines that might improve broad scale biodiversity analyses in the near future. More holistic reconstructions of ecological communities can greatly increase the value of metabarcoding studies, improving understanding of the consequences of ongoing environmental changes on the multiple components of biodiversity.
Topics: Biodiversity; DNA; DNA Barcoding, Taxonomic; DNA Primers; Ecology
PubMed: 36762839
DOI: 10.1111/mec.16881 -
Nucleic Acids Research Aug 2023Human PrimPol possesses DNA primase and DNA polymerase activities and restarts stalled replication forks protecting cells against DNA damage in nuclei and mitochondria....
Human PrimPol possesses DNA primase and DNA polymerase activities and restarts stalled replication forks protecting cells against DNA damage in nuclei and mitochondria. The zinc-binding motif (ZnFn) of the C-terminal domain (CTD) of PrimPol is required for DNA primase activity but the mechanism is not clear. In this work, we biochemically demonstrate that PrimPol initiates de novo DNA synthesis in cis-orientation, when the N-terminal catalytic domain (NTD) and the CTD of the same molecule cooperate for substrates binding and catalysis. The modeling studies revealed that PrimPol uses a similar mode of initiating NTP coordination as the human primase. The ZnFn motif residue Arg417 is required for binding the 5'-triphosphate group that stabilizes the PrimPol complex with a DNA template-primer. We found that the NTD alone is able to initiate DNA synthesis, and the CTD stimulates the primase activity of NTD. The regulatory role of the RPA-binding motif in the modulation of PrimPol binding to DNA is also demonstrated.
Topics: Humans; DNA-Directed DNA Polymerase; DNA Primase; DNA Replication; DNA; DNA Primers; Catalysis; Multifunctional Enzymes
PubMed: 37326028
DOI: 10.1093/nar/gkad507 -
Communications Chemistry Jun 2024Oligonucleotides are advancing as essential materials for the development of new therapeutics, artificial genes, or in storage of information applications. Hitherto, our... (Review)
Review
Oligonucleotides are advancing as essential materials for the development of new therapeutics, artificial genes, or in storage of information applications. Hitherto, our capacity to write (i.e., synthesize) oligonucleotides is not as efficient as that to read (i.e., sequencing) DNA/RNA. Alternative, biocatalytic methods for the de novo synthesis of natural or modified oligonucleotides are in dire need to circumvent the limitations of traditional synthetic approaches. This Perspective article summarizes recent progress made in controlled enzymatic synthesis, where temporary blocked nucleotides are incorporated into immobilized primers by polymerases. While robust protocols have been established for DNA, RNA or XNA synthesis is more challenging. Nevertheless, using a suitable combination of protected nucleotides and polymerase has shown promises to produce RNA oligonucleotides even though the production of long DNA/RNA/XNA sequences (>1000 nt) remains challenging. We surmise that merging ligase- and polymerase-based synthesis would help to circumvent the current shortcomings of controlled enzymatic synthesis.
PubMed: 38890393
DOI: 10.1038/s42004-024-01216-0 -
Cancer Science Jul 2023Lung adenocarcinoma is classified morphologically into five histological subtypes according to the WHO classification. While each histological subtype correlates with a...
Lung adenocarcinoma is classified morphologically into five histological subtypes according to the WHO classification. While each histological subtype correlates with a distinct prognosis, the molecular basis has not been fully elucidated. Here we conducted DNA methylation analysis of 30 lung adenocarcinoma cases annotated with the predominant histological subtypes and three normal lung cases using the Infinium BeadChip. Unsupervised hierarchical clustering analysis revealed three subgroups with different methylation levels: high-, intermediate-, and low-methylation epigenotypes (HME, IME, and LME). Micropapillary pattern (MPP)-predominant cases and those with MPP components were significantly enriched in HME (p = 0.02 and p = 0.03, respectively). HME cases showed a significantly poor prognosis for recurrence-free survival (p < 0.001) and overall survival (p = 0.006). We identified 365 HME marker genes specifically hypermethylated in HME cases with enrichment of "cell morphogenesis" related genes; 305 IME marker genes hypermethylated in HME and IME, but not in LME, with enrichment "embryonic organ morphogenesis"-related genes; 257 Common marker genes hypermethylated commonly in all cancer cases, with enrichment of "regionalization"-related genes. We extracted surrogate markers for each epigenotype and designed pyrosequencing primers for five HME markers (TCERG1L, CXCL12, FAM181B, HOXA11, GAD2), three IME markers (TBX18, ZNF154, NWD2) and three Common markers (SCT, GJD2, BARHL2). DNA methylation profiling using Infinium data was validated by pyrosequencing, and HME cases defined by pyrosequencing results also showed the worse recurrence-free survival. In conclusion, lung adenocarcinomas are stratified into subtypes with distinct DNA methylation levels, and the high-methylation subtype correlated with MPP-predominant cases and those with MPP components and showed a poor prognosis.
Topics: Humans; DNA Methylation; Adenocarcinoma of Lung; Prognosis; Biomarkers; Lung Neoplasms; Neoplasm Staging; Kruppel-Like Transcription Factors
PubMed: 37082886
DOI: 10.1111/cas.15817