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- PDB-8g5k: Cryo-EM structure of the Wedge Alignment Complex (VIII) of Human ... -
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Basic information
Entry | Database: PDB / ID: 8g5k | ||||||
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Title | Cryo-EM structure of the Wedge Alignment Complex (VIII) of Human Mitochondrial DNA Polymerase Gamma | ||||||
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![]() | REPLICATION/DNA / Mitochondrial DNA Polymerase / DNA Proofreading / Wedge alignment / REPLICATION / REPLICATION-DNA complex | ||||||
Function / homology | ![]() gamma DNA polymerase complex / mitochondrial DNA replication / positive regulation of DNA-directed DNA polymerase activity / single-stranded DNA 3'-5' DNA exonuclease activity / Hydrolases; Acting on ester bonds; Exodeoxyribonucleases producing 5'-phosphomonoesters / DNA metabolic process / DNA replication proofreading / DNA polymerase processivity factor activity / mitochondrial nucleoid / Lyases; Carbon-oxygen lyases; Other carbon-oxygen lyases ...gamma DNA polymerase complex / mitochondrial DNA replication / positive regulation of DNA-directed DNA polymerase activity / single-stranded DNA 3'-5' DNA exonuclease activity / Hydrolases; Acting on ester bonds; Exodeoxyribonucleases producing 5'-phosphomonoesters / DNA metabolic process / DNA replication proofreading / DNA polymerase processivity factor activity / mitochondrial nucleoid / Lyases; Carbon-oxygen lyases; Other carbon-oxygen lyases / 5'-deoxyribose-5-phosphate lyase activity / DNA polymerase binding / 3'-5' exonuclease activity / base-excision repair, gap-filling / Transcriptional activation of mitochondrial biogenesis / base-excision repair / DNA-templated DNA replication / protease binding / double-stranded DNA binding / in utero embryonic development / DNA-directed DNA polymerase / DNA-directed DNA polymerase activity / mitochondrial matrix / intracellular membrane-bounded organelle / chromatin binding / protein-containing complex / mitochondrion / DNA binding / identical protein binding / cytoplasm Similarity search - Function | ||||||
Biological species | ![]() synthetic construct (others) | ||||||
Method | ELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.9 Å | ||||||
![]() | Nayak, A.R. / Buchel, G. / Herbine, K.H. / Sarfallah, A. / Sokolova, V.O. / Zamudio-Ochoa, A. / Temiakov, D. | ||||||
Funding support | ![]()
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![]() | ![]() Title: Structural basis for DNA proofreading. Authors: Gina Buchel / Ashok R Nayak / Karl Herbine / Azadeh Sarfallah / Viktoriia O Sokolova / Angelica Zamudio-Ochoa / Dmitry Temiakov / ![]() Abstract: DNA polymerase (DNAP) can correct errors in DNA during replication by proofreading, a process critical for cell viability. However, the mechanism by which an erroneously incorporated base ...DNA polymerase (DNAP) can correct errors in DNA during replication by proofreading, a process critical for cell viability. However, the mechanism by which an erroneously incorporated base translocates from the polymerase to the exonuclease site and the corrected DNA terminus returns has remained elusive. Here, we present an ensemble of nine high-resolution structures representing human mitochondrial DNA polymerase Gamma, Polγ, captured during consecutive proofreading steps. The structures reveal key events, including mismatched base recognition, its dissociation from the polymerase site, forward translocation of DNAP, alterations in DNA trajectory, repositioning and refolding of elements for primer separation, DNAP backtracking, and displacement of the mismatched base into the exonuclease site. Altogether, our findings suggest a conserved 'bolt-action' mechanism of proofreading based on iterative cycles of DNAP translocation without dissociation from the DNA, facilitating primer transfer between catalytic sites. Functional assays and mutagenesis corroborate this mechanism, connecting pathogenic mutations to crucial structural elements in proofreading steps. | ||||||
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Structure visualization
Structure viewer | Molecule: ![]() ![]() |
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Downloads & links
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Download
PDBx/mmCIF format | ![]() | 359.9 KB | Display | ![]() |
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PDB format | ![]() | 277.3 KB | Display | ![]() |
PDBx/mmJSON format | ![]() | Tree view | ![]() | |
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-Validation report
Arichive directory | ![]() ![]() | HTTPS FTP |
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-Related structure data
Related structure data | ![]() 29747MC ![]() 8g5iC ![]() 8g5jC ![]() 8g5lC ![]() 8g5mC ![]() 8g5nC ![]() 8g5oC ![]() 8g5pC ![]() 8t7eC M: map data used to model this data C: citing same article ( |
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Similar structure data | Similarity search - Function & homology ![]() |
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Links
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Assembly
Deposited unit | ![]()
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Components
#1: Protein | Mass: 139730.703 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) ![]() ![]() ![]() | ||||
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#2: Protein | Mass: 54991.000 Da / Num. of mol.: 2 Source method: isolated from a genetically manipulated source Source: (gene. exp.) ![]() ![]() ![]() #3: DNA chain | | Mass: 6209.018 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others) #4: DNA chain | | Mass: 7894.083 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others) |
-Experimental details
-Experiment
Experiment | Method: ELECTRON MICROSCOPY |
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EM experiment | Aggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction |
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Sample preparation
Component | Name: Cryo-EM structure of the Wedge Alignment Complex (VIII) of Human Mitochondrial DNA Polymerase Gamma Type: COMPLEX Details: Human mitochondrial DNA polymerase PolG (wild type) assembled on a DNA-DNA scaffold Entity ID: all / Source: RECOMBINANT |
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Molecular weight | Value: 0.362 MDa / Experimental value: YES |
Source (natural) | Organism: ![]() |
Source (recombinant) | Organism: ![]() ![]() |
Buffer solution | pH: 7.9 Details: 10 mM Tris pH 7.9, 100 mM NaCl, 10 mM DTT, and 2 mM MgCl2 |
Specimen | Conc.: 0.52 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES Details: 2 uM complex of the wild type PolG and DNA-DNA scaffold containing a mismatched base |
Specimen support | Grid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: UltrAuFoil R1.2/1.3 |
Vitrification | Instrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 K |
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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: ![]() |
Electron lens | Mode: BRIGHT FIELD / Nominal magnification: 105000 X / Nominal defocus max: 18000 nm / Nominal defocus min: 500 nm / Cs: 2.7 mm |
Specimen holder | Cryogen: NITROGEN |
Image recording | Electron dose: 70 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) / Num. of grids imaged: 2 / Num. of real images: 10572 |
EM imaging optics | Energyfilter slit width: 20 eV |
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Processing
EM software |
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CTF correction | Type: PHASE FLIPPING AND AMPLITUDE CORRECTION | ||||||||||||||||||||||||
Particle selection | Num. of particles selected: 14667792 | ||||||||||||||||||||||||
Symmetry | Point symmetry: C1 (asymmetric) | ||||||||||||||||||||||||
3D reconstruction | Resolution: 2.9 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 462003 / Num. of class averages: 1 / Symmetry type: POINT | ||||||||||||||||||||||||
Atomic model building | Protocol: FLEXIBLE FIT / Space: REAL | ||||||||||||||||||||||||
Refine LS restraints |
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