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157 results for “DNA Polymerase”
AlphaFold2 models of human DNA polymerase epsilon catalytic subunit A
<p>Models of the structure of human DNA polymerase epsilon catalytic subunit A built with the program AlphaFold2. Files are in mmCIF format. Models include:</p> <p>1) <a href="https://zenodo.org/api/files/c8652c0b-1bc6-44d5-b11e-e8e107dba67e/DPOE1_HUMAN_AF2_NterminalLobe_4m8o.cif">DPOE1_HUMAN_AF2_NterminalLobe_4m8o.cif </a>: AlphaFold2 model built with template PDB:4M8O (yeast DNA polymerase epsilon N-terminal lobe with bound DNA)</p> <p>2) <a href="https://zenodo.org/api/files/c8652c0b-1bc6-44d5-b11e-e8e107dba67e/DPOE1_HUMAN_AF2_FullLength_6wjv.cif">DPOE1_HUMAN_AF2_FullLength_6wjv.cif </a>: AlphaFold2 model built with template PDB:6WJV (yeast DNA polymerase epsilon full-length protein, N and C terminal lobes, without DNA).</p> <p>3) <a href="https://zenodo.org/api/files/c8652c0b-1bc6-44d5-b11e-e8e107dba67e/DPOE1_HUMAN_AF2_NterminalLobe_4m8o_withDNA.cif">DPOE1_HUMAN_AF2_NterminalLobe_4m8o_withDNA.cif</a> : AlphaFold2 model built with template 4M8O (File #1 above) with DNA added from PDB entry 4M8O and subjected to energy minimization with the program AMBER using force field ff14SB.</p> <p>The models were used to estimate the change in free energy of mutations found in patients with ovarian cancer, colon cancer, and endometrial cancer. Paper to be submitted Dec 2022.</p>
Kinetic in support of "Pre-Steady-State Kinetic Characterization of an Antibiotic-Resistant Mutant of Staphylococcus aureus DNA Polymerase PolC"
<p>These are KinTek Explorer mechanism files containing the data and analysis described in the manuscript "Pre-Steady-State Kinetic Characterization of an Antibiotic-Resistant Mutant of Staphylococcus aureus DNA Polymerase PolC" (bioRxiv 2022.10.04.510889; doi: https://doi.org/10.1101/2022.10.04.510889). </p>
Gleaning Euglenozoa-specific DNA polymerases in public single-cell transcriptome data
<p><span>Multiple genes encoding family A DNA polymerases (famA DNAPs), which are evolutionary relatives of DNA polymerase </span><span>I</span><span> (Pol</span><span>I</span><span>) in bacteria and phages, have been found in eukaryotic genomes, and many of these proteins are used mainly in organelles. Among members of the phylum Euglenozoa, distinct types of famA DNAP, Pol</span><span>I</span><span>A, Pol</span><span>I</span><span>BCD+, POP, and eugPolA, have been found. It is intriguing how the suite of famA DNAPs had been established during the evolution of Euglenozoa, but the DNAP data have not been sampled from the taxa that sufficiently represent the diversity of this phylum. In particular, little sequence data were available for basal branching species in Euglenozoa until recently. Thanks to the single-cell transcriptome data from symbiontids and phagotrophic euglenids, we have an opportunity to cover the "hole" in the repertory of famA DNAPs in the deep branches in Euglenozoa. The current study identified 16 new famA DNAP sequences in the transcriptome data from 33 phagotrophic euglenids and two symbiontids, respectively. Based on the new famA DNAP sequences, the updated diversity and evolution of famA DNAPs in Euglenozoa are discussed.</span></p>
Supplementary data for: Encyclopaedia of family A DNA polymerases localized in organelles: Evolutionary contribution of bacteria including the proto-mitochondrion
<p><span>DNA polymerases (DNAPs) synthesize DNA from deoxyribonucleotides in a semi-conservative manner and serve as the core of DNA replication and repair machinery. In eukaryotic cells, there are two genome-containing organelles, mitochondria and plastids, that were derived from an α-proteobacterium and a cyanobacterium, respectively. Except for rare cases of genome-lacking mitochondria and plastids, both organelles must be served by nucleus-encoded DNAPs that localize and work in them to maintain their genomes. The evolution of organellar DNAPs has yet to be fully understood because of two unsettled issues. First, the diversity of organellar DNAPs has not been elucidated in the full spectrum of eukaryotes. Second, it is unclear when the DNAPs that were used originally in the endosymbiotic bacteria giving rise to mitochondria and plastids were discarded, as the organellar DNAPs known to date show no phylogenetic affinity to those of the extant α-proteobacteria or cyanobacteria. </span><span>In this study, we identified from diverse eukaryotes 134</span> <span>family A DNAP sequences, which were classified into 10 novel types, and explored their evolutionary origins. The subcellular localizations of selected DNAPs were further examined experimentally. The results presented here suggest that the diversity of organellar DNAPs has been shaped by multiple transfers of the Pol</span><span>I</span><span> gene from phylogenetically broad bacteria, and their occurrence in eukaryotes was additionally impacted by secondary plastid endosymbioses. Finally, we propose that the last eukaryotic common ancestor may have possessed two mitochondrial DNAPs, POP and a candidate of the direct descendant of the proto-mitochondrial DNAP, rdxPolA, identified in this study.</span></p>
Data for paper: Analysis and control of untemplated DNA polymerase activity for guided synthesis of kilobase-scale DNA sequences
<p>This deposit contains data for the paper entitled: "<strong>Analysis and control of untemplated DNA polymerase activity for guided synthesis of kilobase-scale DNA sequences</strong>"</p> <p>Contents include:</p> <ul> <li><strong>00_sequencing-metadata.xlsx</strong> - Metadata for each sequencing sample.</li> <li><strong>01_raw-fastqs.zip</strong> - Raw basecalled FASTQ data.</li> <li><strong>02_clean-fastas.zip</strong> - Cleaned FASTA files (removal of adapters and barcode sequences).</li> <li><strong>03_read-statistics.zip</strong> - Read statistics for all samples.</li> <li><strong>04_sequence-analysis.zip</strong> - Sequence analysis output for all samples.</li> <li><strong>05_qpcr-data.xlsx</strong> - qPCR data for all the dNTP mixes studied.</li> <li><strong>06_analysis-scripts.zip</strong> - Analysis scripts.</li> </ul>
Dual mechanisms by which DNA polymerase epsilon discriminates ribonucleotides
<p>Topologies, parameters, and molecular dynamics simulation files to run simulations of the wild-type and M644G variants of DNA Polymerase epsilon bound to deoxyadenosine triphosphate (dATP) and adenosine triphosphate (ATP).</p>
Thermally controlled intein splicing of engineered DNA polymerases provides a robust and generalizable solution for accurate and sensitive molecular diagnostics
<p>DNA polymerases are essential for nucleic acid synthesis, cloning, sequencing and molecular diagnostics technologies. Conditional intein splicing is a powerful tool for controlling enzyme reactions. We have engineered a thermal switch into thermostable DNA polymerases from two structurally distinct polymerase families by inserting a thermally activated intein domain into a surface loop that is integral to the polymerase active site, thereby blocking DNA or RNA template access. The fusion proteins are inactive but retain their structures such that the intein excises during a heat pulse delivered at 70–80°C to generate spliced, active polymerases. This straightforward thermal activation step provides a highly effective, one-component 'hot-start' control of PCR reactions that enables accurate target amplification by minimizing unwanted by-products generated by off-target reactions. In one engineered enzyme, derived from <em>Thermus aquaticus</em> DNA polymerase, both DNA polymerase and reverse transcriptase activities are controlled by the intein, enabling single-reagent amplification of DNA and RNA under hot-start conditions. This engineered polymerase provides high-sensitivity detection for molecular diagnostics applications, amplifying 5–6 copies of the tested DNA and RNA targets with >95% certainty. The design principles used to engineer the inteins can be readily applied to construct other conditionally activated nucleic acid processing enzymes.</p>
Thermally controlled intein splicing of engineered DNA polymerases provides a robust and generalizable solution for accurate and sensitive molecular diagnostics
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Gleaning Euglenozoa-specific DNA polymerases in public single-cell transcriptome data
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Supplementary data for: Encyclopaedia of family A DNA polymerases localized in organelles: Evolutionary contribution of bacteria including the proto-mitochondrion
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Phage origin of mitochondrion-localized family A DNA polymerases in kinetoplastids and diplonemids
<p>Mitochondria retain their own genomes as other bacterial endosymbiont-derived organelles. Nevertheless, no protein for DNA replication and repair is encoded in any mitochondrial genomes (mtDNAs) assessed to date, suggesting the nucleus primarily governs the maintenance of mtDNA. As the proteins of diverse evolutionary origins occupy a large proportion of the current mitochondrial proteomes, we anticipate finding the same evolutionary trend in the nucleus-encoded machinery for mtDNA maintenance. Indeed, none of the DNA polymerases (DNAPs) in the mitochondrial endosymbiont, a putative α-proteobacterium, seemingly had been inherited by their descendants (mitochondria), as none of the known types of mitochondrion-localized DNAP showed a specific affinity to the α-proteobacterial DNAPs. Nevertheless, we currently have no concrete idea of how and when the known types of mitochondrion-localized DNAPs emerged. We here explored the origins of mitochondrion-localized DNAPs after the improvement of the samplings of DNAPs from bacteria and phages/viruses. Past studies revealed that a set of mitochondrion-localized DNAPs in kinetoplastids and diplonemids, namely PolIB, PolIC, PolID, PolI-Perk1/2, and PolI-dipl (henceforth designated collectively as "PolIBCD+") have emerged from a single DNAP. In this study, we recovered an intimate connection between PolIBCD+ and the DNAPs found in a particular group of phages. Thus, the common ancestor of kinetoplastids and diplonemids most likely converted a laterally acquired phage DNAP into a mitochondrion-localized DNAP that was ancestral to PolIBCD+. The phage origin of PolIBCD+ hints at a potentially large contribution of proteins acquired via non-vertical processes to the machinery for mtDNA maintenance in kinetoplastids and diplonemids.</p>
Mapping fast DNA polymerase exchange during replication
<h2>Abstract</h2> <p>Despite extensive studies on DNA replication, the exchange mechanisms of DNA polymerase during replication remain unclear. Existing models propose that this exchange is facilitated by protein partners like helicase. Here we present data, employing a combination of mechanical DNA manipulation and single fluorescent protein observation, that reveal DNA polymerase undergoing rapid and autonomous exchange during replication not coordinated by other proteins. The DNA polymerase shows fast unbinding and rebinding dynamics, displaying a preference for either exonuclease or polymerase activity, or pausing events, during each brief binding event. We also observed a 'memory effect' in DNA polymerase rebinding, i.e., the enzyme tends to preserve its prior activity upon reassociation. This effect, potentially linked to the ssDNA/dsDNA junction's conformation, might play a role in regulating binding preference enabling high processivity amidst rapid protein exchange. Taken together, our findings support an autonomous replication model that includes rapid protein exchange, burst of activity, and a 'memory effect' while moving processively forward. </p> <h2>Data Set Description</h2> <p>The study presents findings on the autonomous exchange mechanisms of DNA polymerase at the replication fork. Employing a combination of mechanical DNA manipulation and single fluorescent protein observation, the research reveals rapid and autonomous exchange of DNA polymerase, independent of protein partners like helicase. It highlights the enzyme's preference for exonuclease or polymerase activity and a 'memory effect' during binding events. In this data repo, we provided all the raw data used to reproduce the findings.</p> <h2>Methodology</h2> <p>The single-molecule experiments were performed at room temperature in a 5-channel microfluidic flow cell using the LUMICKS C-Trap instrument that combines dual optical trapping, confocal microscopy, and microfluidics for single-molecule assays. Data analysis was conducted using Python, Origin, and MATLAB. Detailed methodology and instrument specifications are provided in the associated publication.</p> <h2>File Formats</h2> <ul> <li> <p>All data is obtained from C-trap in .tdms format.</p> </li> </ul> <h2>Usage Notes</h2> <p>The custom-written python scripts used in this study is available at <a href="https://github.com/longfuxu/DNAPolymeraseProject">https://github.com/longfuxu/DNAPolymeraseProject</a>, under the MPL-2.0 license. The repository includes example dataset, example Jupiter notebook, along with a detailed README file for instructions on installation and usage.</p> <h2>Acknowledgments</h2> <p>We thank Seyda Aca and Sandrine D'Haene for assistance with protein purification and DNA construction, Noémie Danné for help with implementing the step-fitting algorithm. We thank Erwin Peterman for critical reading and constructive feedbacks of this manuscript. This work was financially supported by a PhD fellowship from China Scholarship Council (To L.X., funding No. 201704910912), the European Union H2020 Marie-Sklowdowska Curie International Training Network AntiHelix (To G.J.L.W., funding No. 859853), and the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation program MONOCHROME (to G.J.L.W., funding No.883240).</p> <h2>Competing Interest</h2> <p>The combined optical tweezers and fluorescence technologies used in this article are patented and licensed to LUMICKS B.V., in which M.T.J.H., and G.J.L.W. declare a financial interest. All other authors declare that they have no competing interests.</p> <h2>Author Contributions</h2> <p>L.X. and G.J.L.W. conceptualized the research. L.X. prepared protein samples and collected single-molecule data and analyzed data; M.T.J.H. analyzed data; L.X., M.T.J.H. and G.J.L.W. wrote and edited the manuscript; G.J.L.W. supervised the project; the manuscript is read, revised, and confirmed by all the listed authors.</p>
Microscopy images of the DNA and RNA polymerase II distribution inside nuclei of cell cultures obtained from pluripotent zebrafish embryos
<p><strong>Image data description</strong></p> <p>Color channels in the image data:</p> <ol> <li>First channel: DNA (Hoechst 33342)</li> <li>Second channel: Pol II Ser2P (Elongating RNA polymerase II, indirect immunofluorescence, STAR RED)</li> <li>Third channel: Pol II Ser5P (Recruited RNA polymerase II, indirect immunofluorescence, Alexa 594)</li> </ol> <p><strong>Sample description</strong></p> <p>Praimary cell cultures were prepared from pluripotent zebrafish embryos were collected at the sphere stage of development, treated with chemical inhibitors of transcription (Control, 500 mM Flavopiridol, 500 mM Triptolide, all for 30 min) and fixed overnight (2% formaldehyde in culutre media, 30 min, room temperature; followed by 8% formaldehyde for 15 min at room temperature). RNA polymerase in the recruited state (Pol II Ser5P) and the elongating state (Pol II Ser2P) were labeled by indirect immunofluorescence (permeabilization 0.5% Triton X-100 in PBS 15 min room temperature, 30 min blocking 4% BSA in PBST, rat IgG anti-Pol II Ser5P & rabbit IgG anti-Pol II Ser2P in 4% BSA in PBST overnight 4°C, anti-rat Alexa 594 & anti-rabbit STAR RED in 4% BSA in PBST overnight 4°C). Mounted in VectaShield H-1000 with 2 µM Hoechst 33342 added for fluorescent DNA labeling. Scan of lab book page is included in the repository.</p> <p>The data set contains images obtained from two samples for each condition.</p> <p><strong>Imaging</strong></p> <p>Microscopy images acquired using VisiTech iSIM with dual camera setup. Objective Nikon CFI SR HP Apo<br> TIRF 100XAC Oil, color channels acquired in a sequence to reduce overlap (DNA + Ser2P acquired simultaneously on two camerase, Ser5P acquired after on a single camera), z-stack settings optimized to ensure reliable xyz alignment of channels. Imags were cropped to the region with best signal and resolution in the DNA channel, same region used throughout the entire dataset. all imaging settings were kept unchanged over the course of acquisition, all images acquired in a single session of 4 hours.</p> <p><strong>Advice for image processing</strong></p> <p>Images can be loaded for processing with the OME bioformats importer. An import script for MatLab is available through the Hilbert lab: https://github.com/lhilbert/NuclearObjects_ImageAnalysis</p> <p><strong>Author contributions</strong></p> <p>AN & MS provided embryos, carried out cell culture, and prepared samples, LH performed microscopy</p>
Starting Coordinates for DNA Polymerase III* WT and Mutants
<p>Included are the starting coordinates and parameters for both MD and QM/MM simulations of DNA Polymerase III* of <em>E. coli</em>. The mutants studied were ß-L82E/L82'E and ß-L82D/L82'D. Systems studied include one with shorter DNA and the other with longer DNA.</p>
Data related to the manuscript "Reduced amplification by phi29 DNA polymerase in the presence of unbound oligos during rolling circle amplification"
<p>Raw data from AC susceptometry and processed data in excel sheets.</p>
The Polymerase Chain Reaction (PCR) Analysis of Nasal Polyps for Fungal DNA
ClinicalTrials.gov study NCT00739921. IPD Sharing: Not stated. Countries: 1. Publications: 3.
Phage origin of mitochondrion-localized family A DNA polymerases in kinetoplastids and diplonemids
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Data from: Structural consequence of the most frequently recurring cancer-associated substitution in DNA polymerase ε
The most frequently recurring cancer-associated DNA polymerase ε (Pol ε) mutation is a P286R substitution in the exonuclease domain. While originally proposed to increase genome instability by disrupting exonucleolytic proofreading, the P286R variant was later found to be significantly more pathogenic than Pol ε proofreading deficiency per se. The mechanisms underlying its stronger impact remained unclear. Here we report the crystal structure of the yeast orthologue, Pol ε−P301R, complexed with DNA and an incoming dNTP. Structural changes in the protein are confined to the exonuclease domain, with R301 pointing towards the exonuclease site. Molecular dynamics simulations suggest that R301 interferes with DNA binding to the exonuclease site, an outcome not observed with the exonuclease-inactive Pol ε−D290A,E292A variant lacking the catalytic residues. These results reveal a distinct mechanism of exonuclease inactivation by the P301R substitution and a likely basis for its dramatically higher mutagenic and tumorigenic effects.
Figure 6 from: Todorova N, Rangelov M, Dincheva I, Badjakov I, Enchev V, Markova N (2022) Potential of hydroxybenzoic acids from Graptopetalum paraguayense for inhibiting of herpes simplex virus DNA polymerase – metabolome profiling, molecular docking and quantum-chemical analysis. Pharmacia 69(1): 113-123. https://doi.org/10.3897/pharmacia.69.e79467
Figure 6 Structures of amino acids from GP phenolic fraction "C", optimized at B3LYP/6-31+G(d,p) level.
Figure 3 from: Todorova N, Rangelov M, Dincheva I, Badjakov I, Enchev V, Markova N (2022) Potential of hydroxybenzoic acids from Graptopetalum paraguayense for inhibiting of herpes simplex virus DNA polymerase – metabolome profiling, molecular docking and quantum-chemical analysis. Pharmacia 69(1): 113-123. https://doi.org/10.3897/pharmacia.69.e79467
Figure 3 Acyclovir triphosphate and its vicinity in the DNA polymerase pocket after docking procedure: a) 3D plane of view and b) 2D plane of view. The interactions of the ligand in the active site cavity are represented as follows: the proximity contour is depicted with a black dotted line; solvent accessibility, as blue clouds around atoms or blue shadows around amino acid residues; polar amino acids are displayed with pink, while the lipophilic ones are in green. Basic amino acids are outlined with blue and the acidic – with red. Hydrogen bond interactions are depicted with dotted arrows, while the ionic ones are depicted with dotted lines.
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Allen Brain Atlas
Allen Brain Atlas is an Allen Institute collection of brain map atlases, datasets, APIs, and analysis tools covering mouse, human, and non-human primate brain resources.
Annotated Behaviour and Observability Dataset (ABODe)
ABODe is a University of Edinburgh DataShare dataset for behavior classification in group-housed mice using home-cage video, identities, bounding boxes, ground-plate positions, and annotator labels.
DANDI Archive for NWB datasets
DANDI is a BRAIN Initiative archive for publishing and sharing neurophysiology data, including electrophysiology, optophysiology, and behavioral data packaged as NWB and related standards.
International Brain Laboratory public data
The International Brain Laboratory public data releases expose standardized mouse decision-making experiments, including Neuropixels recordings, widefield calcium imaging, behavior, and session metadata accessed through the ONE API.
OpenNeuro
OpenNeuro is a free, open platform for sharing neuroimaging datasets, with public search, dataset pages, and download paths for web, S3, DataLad, and the OpenNeuro CLI.