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- PDB-12sl: Structure of a Strand-Displacement Complex of Human Mitochondrial... -

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Basic information

Entry
Database: PDB / ID: 12sl
TitleStructure of a Strand-Displacement Complex of Human Mitochondrial DNA Polymerase Gamma
Components
  • (DNA polymerase subunit gamma- ...) x 2
  • Template DNA
  • primer DNA
KeywordsREPLICATION / Mitochondrial DNA Polymerase Gamma / PolG / DNA strand displacement
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
3'-DEOXYTHYMIDINE-5'-MONOPHOSPHATE / 2'-DEOXYADENOSINE 5'-TRIPHOSPHATE / 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: 2.93 Å
AuthorsNayak, A.R. / Sokolova, V.O. / 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
DepositionApr 16, 2026Deposition 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 DNA
T: Template DNA
hetero molecules


Theoretical massNumber of molelcules
Total (without water)263,7158
Polymers262,8935
Non-polymers8223
Water00
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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DNA chain , 2 types, 2 molecules PT

#3: DNA chain primer DNA


Mass: 5878.771 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#4: DNA chain Template DNA


Mass: 7301.696 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)

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Non-polymers , 3 types, 3 molecules

#5: Chemical ChemComp-DTP / 2'-DEOXYADENOSINE 5'-TRIPHOSPHATE


Mass: 491.182 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C10H16N5O12P3
#6: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Mg
#7: Chemical ChemComp-2DT / 3'-DEOXYTHYMIDINE-5'-MONOPHOSPHATE / 2',3'-DIDEOXYTHYMIDINE-5'-MONOPHOSPHATE


Type: DNA linking / Mass: 306.209 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C10H15N2O7P

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Details

Has ligand of interestN
Has protein modificationY

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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: Strand displacement Complex of Human Mitochondrial DNA Polymerase Gamma
Type: COMPLEX
Details: Strand-displacement complex of human mitochondrial DNA polymerase Gamma (exo-), assembled on a 1-nt gapped primer-template substrate contiguous with a downstream duplex containing a 4-nt 5' ...Details: Strand-displacement complex of human mitochondrial DNA polymerase Gamma (exo-), assembled on a 1-nt gapped primer-template substrate contiguous with a downstream duplex containing a 4-nt 5' non-template flap ss-DNA. The primer terminus was extended by four nucleotides (three dG followed by a chain-terminating ddT), thereby promoting coordinated displacement of four non-template nucleotides from the downstream DNA duplex. The ternary catalytic complex is stabilized by a bound incoming nucleotide (dATP) at the polymerase active site in the presence of 5 mM MgCl2.
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 5 mM MgCl2
SpecimenConc.: 0.52 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Details: 2 uM strand-displacement complex of human mitochondrial DNA polymerase Gamma (exo-), assembled on an equimolar 1-nt gapped primer-template substrate contiguous with a downstream duplex ...Details: 2 uM strand-displacement complex of human mitochondrial DNA polymerase Gamma (exo-), assembled on an equimolar 1-nt gapped primer-template substrate contiguous with a downstream duplex containing a 4-nt single-stranded non-template flap. The primer was extended by four nucleotides; three successive dG at 0.2 mM, followed by incorporation of a chain-terminating ddT at 0.1 mM), thereby driving coordinated displacement of four nucleotides from the downstream DNA duplex. The resulting ternary catalytic complex was further stabilized by binding of an incoming nucleotide (1 mM dATP) at the polymerase active site in the presence of 5 mM MgCl2.
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
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: 1500 nm / Nominal defocus min: 500 nm / Cs: 2.7 mm / C2 aperture diameter: 100 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN
Image recordingAverage exposure time: 2.6 sec. / Electron dose: 60 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 7236
EM imaging opticsEnergyfilter slit width: 20 eV

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Processing

EM software
IDNameVersionCategory
1cryoSPARC4.6particle selection
2Latitudeimage acquisition
4cryoSPARC4.6CTF correction
7Coot0.9.8.5model fitting
9PHENIX2.0-5936model refinement
12cryoSPARC4.6classification
13cryoSPARC4.63D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 5827907
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 2.93 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 395220 / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL
Atomic model buildingPDB-ID: 9PCK

9pck


Accession code: 9PCK / Source name: PDB / Type: experimental model

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