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- PDB-9pck: Human DNA Polymerase Gamma-RNA-DNA Primer-Template Complex with I... -

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Basic information

Entry
Database: PDB / ID: 9pck
TitleHuman DNA Polymerase Gamma-RNA-DNA Primer-Template Complex with Incoming ATP
Components
  • (DNA polymerase subunit gamma- ...) x 2
  • Template DNA
  • primer RNA
KeywordsREPLICATION / Mitochondrial DNA Polymerase / PolG / Transferase-RNA complex
Function / homology
Function and homology information


gamma DNA polymerase complex / mitochondrial chromosome / positive regulation of DNA-directed DNA polymerase activity / Strand-asynchronous mitochondrial DNA replication / mitochondrial DNA replication / 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 ...gamma DNA polymerase complex / mitochondrial chromosome / positive regulation of DNA-directed DNA polymerase activity / Strand-asynchronous mitochondrial DNA replication / mitochondrial DNA replication / 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 / Lyases; Carbon-oxygen lyases; Other carbon-oxygen lyases / mitochondrial nucleoid / 5'-deoxyribose-5-phosphate lyase activity / base-excision repair, gap-filling / DNA polymerase binding / 3'-5' exonuclease activity / DNA polymerase activity / Transcriptional activation of mitochondrial biogenesis / DNA-templated DNA replication / base-excision repair / protease binding / double-stranded DNA binding / DNA-directed DNA polymerase / DNA-directed DNA polymerase activity / mitochondrial matrix / chromatin binding / protein-containing complex / mitochondrion / DNA binding / identical protein binding
Similarity search - Function
DNA-directed DNA-polymerase, family A, mitochondria / DNA mitochondrial polymerase, exonuclease domain / POLG2, C-terminal / : / DNA mitochondrial polymerase exonuclease domain / Glycyl-tRNA synthetase/DNA polymerase subunit gamma-2 / Anticodon-binding / Anticodon binding domain / Anticodon-binding domain superfamily / DNA-directed DNA polymerase, family A, conserved site ...DNA-directed DNA-polymerase, family A, mitochondria / DNA mitochondrial polymerase, exonuclease domain / POLG2, C-terminal / : / DNA mitochondrial polymerase exonuclease domain / Glycyl-tRNA synthetase/DNA polymerase subunit gamma-2 / Anticodon-binding / Anticodon binding domain / Anticodon-binding domain superfamily / 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 / 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
2',3'-DIDEOXYADENOSINE-5'-MONOPHOSPHATE / THYMIDINE-5'-TRIPHOSPHATE / DNA / DNA (> 10) / RNA / RNA (> 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: 2.6 Å
AuthorsNayak, A.R. / Buchel, G. / 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: Nucleic Acids Res / Year: 2026
Title: Molecular and structural basis for replication initiation and strand separation by human mitochondrial DNA polymerase γ.
Authors: Viktoriia Sokolova / Gina Buchel / Sarah Strock / Ashok R Nayak / Dmitry Temiakov /
Abstract: Defects in human mitochondrial DNA (mtDNA) replication can lead to somatic mutations associated with a range of devastating mitochondrial diseases. However, the molecular mechanisms governing the ...Defects in human mitochondrial DNA (mtDNA) replication can lead to somatic mutations associated with a range of devastating mitochondrial diseases. However, the molecular mechanisms governing the earliest steps of mtDNA replication and their fidelity remain poorly understood. Here, we found that DNA polymerase gamma (Polγ) forms stable complexes with RNA-DNA primer-template substrates, exhibiting greater stability and lower misincorporation than on DNA-primed substrates. Structural analysis revealed that Polγ interacts with the 2'-OH groups of ribose within the first four nucleotides of the primer, explaining the stability of complexes that utilize RNA primers. Although Polγ requires TWINKLE to extend RNA primers, its intrinsic strand-displacement activity allows it to extend DNA primers independently. Structural data further show that the strand-separation mechanism in human Polγ is distinct from that of its yeast paralog, Mip1, and involves previously unresolved elements-the catcher and a GP loop in the exonuclease domain-that support intrinsic strand-displacement synthesis by Polγ. Structure-guided mutagenesis of elements involved in strand separation supports these structural observations. Together, our study provides mechanistic insight into mtDNA replication initiation and strand separation and has implications for understanding the molecular basis of mitochondrial disease.
History
DepositionJun 27, 2025Deposition site: RCSB / Processing site: RCSB
Revision 1.0Oct 7, 2026Provider: repository / Type: Initial release
Revision 1.0Oct 7, 2026Data content type: EM metadata / Data content type: EM metadata / Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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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: primer RNA
T: Template DNA
hetero molecules


Theoretical massNumber of molelcules
Total (without water)262,48910
Polymers261,6195
Non-polymers8705
Water362
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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DNA polymerase subunit gamma- ... , 2 types, 3 molecules ABC

#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: 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 / Plasmid: pProEX / Production host: Escherichia coli (E. coli) / Strain (production host): BL21 Codonplus (DE3) -RIPL / References: UniProt: Q9UHN1, DNA-directed DNA polymerase

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RNA chain / DNA chain , 2 types, 2 molecules PT

#3: RNA chain primer RNA


Mass: 5473.317 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#4: DNA chain Template DNA


Mass: 6433.162 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)

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Non-polymers , 4 types, 7 molecules

#5: Chemical ChemComp-TTP / THYMIDINE-5'-TRIPHOSPHATE


Mass: 482.168 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C10H17N2O14P3 / Feature type: SUBJECT OF INVESTIGATION
#6: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: Mg / Feature type: SUBJECT OF INVESTIGATION
#7: Chemical ChemComp-2DA / 2',3'-DIDEOXYADENOSINE-5'-MONOPHOSPHATE


Type: DNA linking / Mass: 315.222 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C10H14N5O5P / Feature type: SUBJECT OF INVESTIGATION
#8: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Formula: H2O

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Details

Has ligand of interestY
Has protein modificationN

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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 Catalytic Complex of Human Mitochondrial DNA Polymerase Gamma with an RNA-DNA Primer-Template Heteroduplex
Type: COMPLEX
Details: Ternary complex of Human mitochondrial DNA polymerase PolG (Exo-), RNA-DNA primer template, a primer chain terminating nucleotide 2',3'-dideoxyadenosine-5'-monophosphate(2DA), and an ...Details: Ternary complex of Human mitochondrial DNA polymerase PolG (Exo-), RNA-DNA primer template, a primer chain terminating nucleotide 2',3'-dideoxyadenosine-5'-monophosphate(2DA), and an incoming nucleotide thymidine-5'-triphosphate(TTP).
Entity ID: #1-#4 / 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-Hcl 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 exonuclease deficient PolG, and RNA-DNA primer template, chain terminated with dideoxy ATP, with an incoming nucleotide (dTTP).
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: TFS KRIOS / Details: Objective aperture of 100 um was used
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 105000 X / Nominal defocus max: 1800 nm / Nominal defocus min: 400 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: 12703
EM imaging opticsEnergyfilter slit width: 20 eV

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Processing

EM software
IDNameVersionCategory
1cryoSPARC4.4particle selection
2EPUimage acquisition
12cryoSPARC4.43D reconstruction
19PHENIX1.21rc1_5127model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 2.6 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 1353452 / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL

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