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- EMDB-76732: Structure of a Strand-Displacement Complex of Human Mitochondrial... -

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Basic information

Entry
Database: EMDB / ID: EMD-76732
TitleStructure of a Strand-Displacement Complex of Human Mitochondrial DNA Polymerase Gamma
Map dataComposite focused map
Sample
  • Complex: Strand displacement Complex of Human Mitochondrial DNA Polymerase Gamma
    • Protein or peptide: DNA polymerase subunit gamma-1
    • Protein or peptide: DNA polymerase subunit gamma-2, mitochondrial
    • DNA: primer DNA
    • DNA: Template DNA
  • Ligand: 2'-DEOXYADENOSINE 5'-TRIPHOSPHATE
  • Ligand: MAGNESIUM ION
  • Ligand: 3'-DEOXYTHYMIDINE-5'-MONOPHOSPHATE
KeywordsMitochondrial DNA Polymerase Gamma / PolG / DNA strand displacement / REPLICATION
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
DNA polymerase subunit gamma-1 / DNA polymerase subunit gamma-2
Similarity search - Component
Biological speciesHomo sapiens (human) / synthetic construct (others)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.93 Å
AuthorsNayak AR / Sokolova VO / Temiakov D / Sarfallah A / Zamudio-Ochoa A
Funding support United States, 1 items
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, 2026-
Header (metadata) releaseOct 7, 2026-
Map releaseOct 7, 2026-
UpdateOct 7, 2026-
Current statusOct 7, 2026Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_76732.png

Downloads & links

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Map

FileDownload / File: emd_76732.map.gz / Format: CCP4 / Size: 129.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationComposite focused map
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.86 Å/pix.
x 324 pix.
= 277.214 Å		0.86 Å/pix.
x 324 pix.
= 277.214 Å		0.86 Å/pix.
x 324 pix.
= 277.214 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.8556 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.0186
Minimum - Maximum-0.05937224 - 0.18326668
Average (Standard dev.)0.0005065104 (±0.0047237226)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions324324324
Spacing324324324
CellA=B=C: 277.2144 Å
α=β=γ: 90.0 °

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Supplemental data

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Sample components

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Entire : Strand displacement Complex of Human Mitochondrial DNA Polymerase...

EntireName: Strand displacement Complex of Human Mitochondrial DNA Polymerase Gamma
Components
  • Complex: Strand displacement Complex of Human Mitochondrial DNA Polymerase Gamma
    • Protein or peptide: DNA polymerase subunit gamma-1
    • Protein or peptide: DNA polymerase subunit gamma-2, mitochondrial
    • DNA: primer DNA
    • DNA: Template DNA
  • Ligand: 2'-DEOXYADENOSINE 5'-TRIPHOSPHATE
  • Ligand: MAGNESIUM ION
  • Ligand: 3'-DEOXYTHYMIDINE-5'-MONOPHOSPHATE

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Supramolecule #1: Strand displacement Complex of Human Mitochondrial DNA Polymerase...

SupramoleculeName: Strand displacement Complex of Human Mitochondrial DNA Polymerase Gamma
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#4
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.
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 362 KDa

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Macromolecule #1: DNA polymerase subunit gamma-1

MacromoleculeName: DNA polymerase subunit gamma-1 / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO / EC number: DNA-directed DNA polymerase
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 139.730703 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: MSRLLWRKVA GATVGPGPVP APGRWVSSSV PASDPSDGQR RRQQQQQQQQ QQQQQPQQPQ VLSSEGGQLR HNPLDIQMLS RGLHEQIFG QGGEMPGEAA VRRSVEHLQK HGLWGQPAVP LPDVELRLPP LYGDNLDQHF RLLAQKQSLP YLEAANLLLQ A QLPPKPPA ...String:
MSRLLWRKVA GATVGPGPVP APGRWVSSSV PASDPSDGQR RRQQQQQQQQ QQQQQPQQPQ VLSSEGGQLR HNPLDIQMLS RGLHEQIFG QGGEMPGEAA VRRSVEHLQK HGLWGQPAVP LPDVELRLPP LYGDNLDQHF RLLAQKQSLP YLEAANLLLQ A QLPPKPPA WAWAEGWTRY GPEGEAVPVA IPEERALVFD VEVCLAEGTC PTLAVAISPS AWYSWCSQRL VEERYSWTSQ LS PADLIPL EVPTGASSPT QRDWQEQLVV GHNVSFDRAH IREQYLIQGS RMRFLDTMSM HMAISGLSSF QRSLWIAAKQ GKH KVQPPT KQGQKSQRKA RRGPAISSWD WLDISSVNSL AEVHRLYVGG PPLEKEPREL FVKGTMKDIR ENFQDLMQYC AQDV WATHE VFQQQLPLFL ERCPHPVTLA GMLEMGVSYL PVNQNWERYL AEAQGTYEEL QREMKKSLMD LANDACQLLS GERYK EDPW LWDLEWDLQE FKQKKAKKVK KEPATASKLP IEGAGAPGDP MDQEDLGPCS EEEEFQQDVM ARACLQKLKG TTELLP KRP QHLPGHPGWY RKLCPRLDDP AWTPGPSLLS LQMRVTPKLM ALTWDGFPLH YSERHGWGYL VPGRRDNLAK LPTGTTL ES AGVVCPYRAI ESLYRKHCLE QGKQQLMPQE AGLAEEFLLT DNSAIWQTVE ELDYLEVEAE AKMENLRAAV PGQPLALT A RGGPKDTQPS YHHGNGPYND VDIPGCWFFK LPHKDGNSCN VGSPFAKDFL PKMEDGTLQA GPGGASGPRA LEINKMISF WRNAHKRISS QMVVWLPRSA LPRAVIRHPD YDEEGLYGAI LPQVVTAGTI TRRAVEPTWL TASNARPDRV GSELKAMVQA PPGYTLVGA DVDSQELWIA AVLGDAHFAG MHGCTAFGWM TLQGRKSRGT DLHSKTATTV GISREHAKIF NYGRIYGAGQ P FAERLLMQ FNHRLTQQEA AEKAQQMYAA TKGLRWYRLS DEGEWLVREL NLPVDRTEGG WISLQDLRKV QRETARKSQW KK WEVVAER AWKGGTESEM FNKLESIATS DIPRTPVLGC CISRALEPSA VQEEFMTSRV NWVVQSSAVD YLHLMLVAMK WLF EEFAID GRFCISIHDE VRYLVREEDR YRAALALQIT NLLTRCMFAY KLGLNDLPQS VAFFSAVDID RCLRKEVTMD CKTP SNPTG MERRYGIPQG EALDIYQIIE LTKGSLEKRS QPGP

UniProtKB: DNA polymerase subunit gamma-1

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Macromolecule #2: DNA polymerase subunit gamma-2, mitochondrial

MacromoleculeName: DNA polymerase subunit gamma-2, mitochondrial / type: protein_or_peptide / ID: 2 / Number of copies: 2 / Enantiomer: LEVO / EC number: DNA-directed DNA polymerase
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 54.991 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MRSRVAVRAC HKVCRCLLSG FGGRVDAGQP ELLTERSSPK GGHVKSHAEL EGNGEHPEAP GSGEGSEALL EICQRRHFLS GSKQQLSRD SLLSGCHPGF GPLGVELRKN LAAEWWTSVV VFREQVFPVD ALHHKPGPLL PGDSAFRLVS AETLREILQD K ELSKEQLV ...String:
MRSRVAVRAC HKVCRCLLSG FGGRVDAGQP ELLTERSSPK GGHVKSHAEL EGNGEHPEAP GSGEGSEALL EICQRRHFLS GSKQQLSRD SLLSGCHPGF GPLGVELRKN LAAEWWTSVV VFREQVFPVD ALHHKPGPLL PGDSAFRLVS AETLREILQD K ELSKEQLV AFLENVLKTS GKLRENLLHG ALEHYVNCLD LVNKRLPYGL AQIGVCFHPV FDTKQIRNGV KSIGEKTEAS LV WFTPPRT SNQWLDFWLR HRLQWWRKFA MSPSNFSSSD CQDEEGRKGN KLYYNFPWGK ELIETLWNLG DHELLHMYPG NVS KLHGRD GRKNVVPCVL SVNGDLDRGM LAYLYDSFQL TENSFTRKKN LHRKVLKLHP CLAPIKVALD VGRGPTLELR QVCQ GLFNE LLENGISVWP GYLETMQSSL EQLYSKYDEM SILFTVLVTE TTLENGLIHL RSRDTTMKEM MHISKLKDFL IKYIS SAKN V

UniProtKB: DNA polymerase subunit gamma-2

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Macromolecule #3: primer DNA

MacromoleculeName: primer DNA / type: dna / ID: 3 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 5.878771 KDa
SequenceString:
(DC)(DA)(DG)(DT)(DC)(DT)(DG)(DC)(DG)(DG) (DC)(DG)(DC)(DG)(DC)(DG)(DG)(DG)(DT)

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Macromolecule #4: Template DNA

MacromoleculeName: Template DNA / type: dna / ID: 4 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 7.301696 KDa
SequenceString:
(DC)(DG)(DA)(DC)(DT)(DA)(DC)(DC)(DC)(DG) (DC)(DG)(DC)(DG)(DC)(DC)(DG)(DC)(DA)(DG) (DA)(DC)(DT)(DG)

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Macromolecule #5: 2'-DEOXYADENOSINE 5'-TRIPHOSPHATE

MacromoleculeName: 2'-DEOXYADENOSINE 5'-TRIPHOSPHATE / type: ligand / ID: 5 / Number of copies: 1 / Formula: DTP
Molecular weightTheoretical: 491.182 Da
Chemical component information

ChemComp-DTP:
2'-DEOXYADENOSINE 5'-TRIPHOSPHATE

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Macromolecule #6: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 6 / Number of copies: 1 / Formula: MG
Molecular weightTheoretical: 24.305 Da

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Macromolecule #7: 3'-DEOXYTHYMIDINE-5'-MONOPHOSPHATE

MacromoleculeName: 3'-DEOXYTHYMIDINE-5'-MONOPHOSPHATE / type: ligand / ID: 7 / Number of copies: 1 / Formula: 2DT
Molecular weightTheoretical: 306.209 Da
Chemical component information

ChemComp-2DT:
3'-DEOXYTHYMIDINE-5'-MONOPHOSPHATE

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration0.52 mg/mL
BufferpH: 7.9
Details: 10 mM Tris-Hcl pH 7.9, 100 mM Nacl, 10 mM DTT, and 5 mM MgCl2
GridModel: UltrAuFoil R1.2/1.3 / Material: GOLD / Mesh: 300 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Atmosphere: AIR
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV
Details2 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.

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Electron microscopy

MicroscopeTFS KRIOS
Specialist opticsEnergy filter - Slit width: 20 eV
SoftwareName: Latitude
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Number grids imaged: 1 / Number real images: 7236 / Average exposure time: 2.6 sec. / Average electron dose: 60.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 100.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 1.5 µm / Nominal defocus min: 0.5 µm / Nominal magnification: 105000
Sample stageCooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 5827907
CTF correctionSoftware - Name: cryoSPARC (ver. 4.6) / Type: PHASE FLIPPING AND AMPLITUDE CORRECTION
Startup modelType of model: NONE / Details: Ab-initio reconstruction in cryoSPARC
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 2.93 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 4.6) / Number images used: 395220
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
Final 3D classificationNumber classes: 4 / Software - Name: cryoSPARC (ver. 4.6)
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

9pck


Chain - Source name: PDB / Chain - Initial model type: experimental model
SoftwareName: Coot (ver. 0.9.8.5)
RefinementSpace: REAL / Protocol: FLEXIBLE FIT
Output model

PDB-12sl:
Structure of a Strand-Displacement Complex of Human Mitochondrial DNA Polymerase Gamma

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