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- PDB-8g5l: Cryo-EM structure of the Primer Separation Complex (IX) of Human ... -

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Basic information

Entry
Database: PDB / ID: 8g5l
TitleCryo-EM structure of the Primer Separation Complex (IX) of Human Mitochondrial DNA Polymerase Gamma
Components
  • DNA polymerase subunit gamma-1
  • DNA polymerase subunit gamma-2, mitochondrial
  • Mismatched Primer DNA
  • Template DNA
KeywordsREPLICATION/DNA / Mitochondrial DNA Polymerase / DNA Proofreading / Primer Separation / REPLICATION / REPLICATION-DNA complex
Function / homology
Function and homology information


gamma DNA polymerase complex / mitochondrial DNA replication / positive regulation of DNA-directed DNA polymerase activity / DNA replication proofreading / single-stranded DNA 3'-5' DNA exonuclease activity / Hydrolases; Acting on ester bonds; Exodeoxyribonucleases producing 5'-phosphomonoesters / DNA metabolic process / 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 / DNA replication proofreading / single-stranded DNA 3'-5' DNA exonuclease activity / Hydrolases; Acting on ester bonds; Exodeoxyribonucleases producing 5'-phosphomonoesters / DNA metabolic process / DNA polymerase processivity factor activity / mitochondrial nucleoid / Lyases; Carbon-oxygen lyases; Other carbon-oxygen lyases / 5'-deoxyribose-5-phosphate lyase activity / DNA polymerase binding / base-excision repair, gap-filling / 3'-5' exonuclease activity / Transcriptional activation of mitochondrial biogenesis / DNA-templated DNA replication / double-stranded DNA binding / in utero embryonic development / protease binding / 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
DNA-directed DNA-polymerase, family A, mitochondria / DNA mitochondrial polymerase, exonuclease domain / : / DNA mitochondrial polymerase exonuclease domain / POLG2, C-terminal / Glycyl-tRNA synthetase/DNA polymerase subunit gamma-2 / Anticodon-binding / Anticodon binding domain / DNA-directed DNA polymerase, family A, conserved site / DNA polymerase family A signature. ...DNA-directed DNA-polymerase, family A, mitochondria / DNA mitochondrial polymerase, exonuclease domain / : / DNA mitochondrial polymerase exonuclease domain / POLG2, C-terminal / Glycyl-tRNA synthetase/DNA polymerase subunit gamma-2 / Anticodon-binding / Anticodon binding domain / DNA-directed DNA polymerase, family A, conserved site / DNA polymerase family A signature. / DNA-directed DNA polymerase, family A, palm domain / DNA polymerase A domain / Anticodon-binding domain superfamily / Class II Aminoacyl-tRNA synthetase/Biotinyl protein ligase (BPL) and lipoyl protein ligase (LPL) / Ribonuclease H-like superfamily / DNA/RNA polymerase superfamily
Similarity search - Domain/homology
DNA / DNA (> 10) / DNA polymerase subunit gamma-1 / DNA polymerase subunit gamma-2
Similarity search - Component
Biological speciesHomo sapiens (human)
synthetic construct (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3 Å
AuthorsNayak, A.R. / Buchel, G. / Herbine, K.H. / Sarfallah, A. / Sokolova, V.O. / Zamudio-Ochoa, A. / Temiakov, D.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)NIH GM131832 United States
CitationJournal: Nat Commun / Year: 2023
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.
History
DepositionFeb 13, 2023Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jan 10, 2024Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: DNA polymerase subunit gamma-1
B: DNA polymerase subunit gamma-2, mitochondrial
C: DNA polymerase subunit gamma-2, mitochondrial
P: Mismatched Primer DNA
T: Template DNA


Theoretical massNumber of molelcules
Total (without water)263,8165
Polymers263,8165
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein DNA polymerase subunit gamma-1 / / Mitochondrial DNA polymerase catalytic subunit / PolG-alpha


Mass: 139730.703 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: POLG, MDP1, POLG1, POLGA / Plasmid: BACMID / Details (production host): pFastBac1 / Cell line (production host): Sf9 / Production host: Spodoptera frugiperda (fall armyworm) / Tissue (production host): Ovary / References: UniProt: P54098, DNA-directed DNA polymerase
#2: Protein DNA polymerase subunit gamma-2, mitochondrial / / DNA polymerase gamma accessory 55 kDa subunit / p55 / Mitochondrial DNA polymerase accessory ...DNA polymerase gamma accessory 55 kDa subunit / p55 / Mitochondrial DNA polymerase accessory subunit / MtPolB / PolG-beta


Mass: 54991.000 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: POLG2, MTPOLB / Production host: Escherichia coli (E. coli) / Strain (production host): BL21 Codonplus (DE3) - RIPL / References: UniProt: Q9UHN1, DNA-directed DNA polymerase
#3: DNA chain Mismatched Primer DNA


Mass: 6209.018 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#4: DNA chain Template DNA


Mass: 7894.083 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: Cryo-EM structure of the Primer Separation Complex (IX) 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
Molecular weightValue: 0.362 MDa / Experimental value: YES
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Spodoptera frugiperda (fall armyworm)
Buffer solutionpH: 7.9
Details: 10 mM Tris pH 7.9, 100 mM NaCl, 10 mM DTT, and 2 mM MgCl2
SpecimenConc.: 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 supportGrid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: UltrAuFoil R1.2/1.3
VitrificationInstrument: 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
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 105000 X / Nominal defocus max: 18000 nm / Nominal defocus min: 500 nm / Cs: 2.7 mm
Specimen holderCryogen: NITROGEN
Image recordingElectron 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 opticsEnergyfilter slit width: 20 eV

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Processing

EM software
IDNameVersionCategory
1cryoSPARC3.2particle selection
2SerialEMimage acquisition
4CTFFIND4CTF correction
7Coot0.9.8.5model fitting
9PHENIX1.20.1-4487model refinement
12cryoSPARC3.2classification
13cryoSPARC3.23D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 14667792
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 282618 / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00812980
ELECTRON MICROSCOPYf_angle_d0.89917843
ELECTRON MICROSCOPYf_dihedral_angle_d16.2032123
ELECTRON MICROSCOPYf_chiral_restr0.0422004
ELECTRON MICROSCOPYf_plane_restr0.0052196

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