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Yorodumi- PDB-8efk: Structure of Lates calcarifer DNA polymerase theta polymerase dom... -
+Open data
-Basic information
Entry | Database: PDB / ID: 8efk | |||||||||
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Title | Structure of Lates calcarifer DNA polymerase theta polymerase domain with hairpin DNA | |||||||||
Components |
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Keywords | DNA BINDING PROTEIN/DNA / DNA double-strand break repair / Microhomology-mediated end joining / DNA BINDING PROTEIN / DNA BINDING PROTEIN-DNA complex | |||||||||
Function / homology | 2',3'-dideoxyadenosine triphosphate / DNA / DNA (> 10) Function and homology information | |||||||||
Biological species | Lates calcarifer (barramundi perch) | |||||||||
Method | ELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3 Å | |||||||||
Authors | Li, C. / Zhu, H. / Sun, J. / Gao, Y. | |||||||||
Funding support | United States, 2items
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Citation | Journal: Nucleic Acids Res / Year: 2023 Title: Structural basis of DNA polymerase θ mediated DNA end joining. Authors: Chuxuan Li / Hanwen Zhu / Shikai Jin / Leora M Maksoud / Nikhil Jain / Ji Sun / Yang Gao / Abstract: DNA polymerase θ (Pol θ) plays an essential role in the microhomology-mediated end joining (MMEJ) pathway for repairing DNA double-strand breaks. However, the mechanisms by which Pol θ recognizes ...DNA polymerase θ (Pol θ) plays an essential role in the microhomology-mediated end joining (MMEJ) pathway for repairing DNA double-strand breaks. However, the mechanisms by which Pol θ recognizes microhomologous DNA ends and performs low-fidelity DNA synthesis remain unclear. Here, we present cryo-electron microscope structures of the polymerase domain of Lates calcarifer Pol θ with long and short duplex DNA at up to 2.4 Å resolution. Interestingly, Pol θ binds to long and short DNA substrates similarly, with extensive interactions around the active site. Moreover, Pol θ shares a similar active site as high-fidelity A-family polymerases with its finger domain well-closed but differs in having hydrophilic residues surrounding the nascent base pair. Computational simulations and mutagenesis studies suggest that the unique insertion loops of Pol θ help to stabilize short DNA binding and assemble the active site for MMEJ repair. Taken together, our results illustrate the structural basis of Pol θ-mediated MMEJ. | |||||||||
History |
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-Structure visualization
Structure viewer | Molecule: MolmilJmol/JSmol |
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-Downloads & links
-Download
PDBx/mmCIF format | 8efk.cif.gz | 135.6 KB | Display | PDBx/mmCIF format |
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PDB format | pdb8efk.ent.gz | 96.1 KB | Display | PDB format |
PDBx/mmJSON format | 8efk.json.gz | Tree view | PDBx/mmJSON format | |
Others | Other downloads |
-Validation report
Summary document | 8efk_validation.pdf.gz | 1.2 MB | Display | wwPDB validaton report |
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Full document | 8efk_full_validation.pdf.gz | 1.2 MB | Display | |
Data in XML | 8efk_validation.xml.gz | 29.4 KB | Display | |
Data in CIF | 8efk_validation.cif.gz | 41 KB | Display | |
Arichive directory | https://data.pdbj.org/pub/pdb/validation_reports/ef/8efk ftp://data.pdbj.org/pub/pdb/validation_reports/ef/8efk | HTTPS FTP |
-Related structure data
Related structure data | 28084MC 8ef9C 8efcC M: map data used to model this data C: citing same article (ref.) |
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Similar structure data | Similarity search - Function & homologyF&H Search |
-Links
-Assembly
Deposited unit |
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1 |
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-Components
#1: Protein | Mass: 95674.117 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Lates calcarifer (barramundi perch) Production host: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria) |
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#2: DNA chain | Mass: 7278.689 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Lates calcarifer (barramundi perch) |
#3: Chemical | ChemComp-MG / |
#4: Chemical | ChemComp-DDS / |
Has ligand of interest | Y |
-Experimental details
-Experiment
Experiment | Method: ELECTRON MICROSCOPY |
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EM experiment | Aggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction |
-Sample preparation
Component | Name: Ternary complex of Lates calcarifer DNA polymerase Theta with duplex DNA and incoming nucleotide Type: COMPLEX / Entity ID: #1-#2 / Source: MULTIPLE SOURCES |
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Source (natural) | Organism: Lates calcarifer (barramundi perch) |
Source (recombinant) | Organism: Escherichia coli (E. coli) |
Buffer solution | pH: 7.6 |
Specimen | Conc.: 0.8 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES |
Vitrification | Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 295 K |
-Electron microscopy imaging
Experimental equipment | Model: Titan Krios / Image courtesy: FEI Company |
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Microscopy | Model: FEI TITAN KRIOS |
Electron gun | Electron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM |
Electron lens | Mode: BRIGHT FIELD / Nominal defocus max: 1600 nm / Nominal defocus min: 800 nm |
Image recording | Electron dose: 1.2 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) |
-Processing
Software | Name: PHENIX / Version: 1.19.2_4158: / Classification: refinement | ||||||||||||||||||||||||
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CTF correction | Type: PHASE FLIPPING AND AMPLITUDE CORRECTION | ||||||||||||||||||||||||
3D reconstruction | Resolution: 3 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 288920 / Symmetry type: POINT | ||||||||||||||||||||||||
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