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- EMDB-71503: Human DNA Polymerase Gamma-RNA-DNA Primer-Template Complex with I... -

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Entry
Database: EMDB / ID: EMD-71503
TitleHuman DNA Polymerase Gamma-RNA-DNA Primer-Template Complex with Incoming ATP
Map data
Sample
  • Complex: Cryo-EM Structure of the Catalytic Complex of Human Mitochondrial DNA Polymerase Gamma with an RNA-DNA Primer-Template Heteroduplex
    • Protein or peptide: DNA polymerase subunit gamma-1
    • Protein or peptide: DNA polymerase subunit gamma-2, mitochondrial
    • RNA: primer RNA
    • DNA: Template DNA
  • Ligand: THYMIDINE-5'-TRIPHOSPHATE
  • Ligand: MAGNESIUM ION
  • Ligand: 2',3'-DIDEOXYADENOSINE-5'-MONOPHOSPHATE
  • Ligand: water
KeywordsMitochondrial DNA Polymerase / PolG / Transferase-RNA complex / 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: 2.6 Å
AuthorsNayak AR / Buchel G / Temiakov D / Sarfallah A / Sokolova VO / 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
DepositionJun 27, 2025-
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_71503.png

Downloads & links

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Map

FileDownload / File: emd_71503.map.gz / Format: CCP4 / Size: 216 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

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AxesZ (Sec.)Y (Row.)X (Col.)
0.68 Å/pix.
x 384 pix.
= 262.502 Å		0.68 Å/pix.
x 384 pix.
= 262.502 Å		0.68 Å/pix.
x 384 pix.
= 262.502 Å

Surface

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Images are generated by Spider.

Voxel sizeX=Y=Z: 0.6836 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.03
Minimum - Maximum-0.22491837 - 0.41507506
Average (Standard dev.)0.00011989064 (±0.009501796)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions384384384
Spacing384384384
CellA=B=C: 262.5024 Å
α=β=γ: 90.0 °

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Supplemental data

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Additional map: #1

Fileemd_71503_additional_1.map
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Additional map: #2

Fileemd_71503_additional_2.map
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Half map: #2

Fileemd_71503_half_map_1.map
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Half map: #1

Fileemd_71503_half_map_2.map
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Sample components

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Entire : Cryo-EM Structure of the Catalytic Complex of Human Mitochondrial...

EntireName: Cryo-EM Structure of the Catalytic Complex of Human Mitochondrial DNA Polymerase Gamma with an RNA-DNA Primer-Template Heteroduplex
Components
  • Complex: Cryo-EM Structure of the Catalytic Complex of Human Mitochondrial DNA Polymerase Gamma with an RNA-DNA Primer-Template Heteroduplex
    • Protein or peptide: DNA polymerase subunit gamma-1
    • Protein or peptide: DNA polymerase subunit gamma-2, mitochondrial
    • RNA: primer RNA
    • DNA: Template DNA
  • Ligand: THYMIDINE-5'-TRIPHOSPHATE
  • Ligand: MAGNESIUM ION
  • Ligand: 2',3'-DIDEOXYADENOSINE-5'-MONOPHOSPHATE
  • Ligand: water

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Supramolecule #1: Cryo-EM Structure of the Catalytic Complex of Human Mitochondrial...

SupramoleculeName: Cryo-EM Structure of the Catalytic Complex of Human Mitochondrial DNA Polymerase Gamma with an RNA-DNA Primer-Template Heteroduplex
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#4
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).
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 RNA

MacromoleculeName: primer RNA / type: rna / ID: 3 / Number of copies: 1
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 5.473317 KDa
SequenceString:
GACAGUCUGC GGCGCGC

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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: 6.433162 KDa
SequenceString:
(DA)(DA)(DA)(DT)(DG)(DC)(DG)(DC)(DG)(DC) (DC)(DG)(DC)(DA)(DG)(DA)(DC)(DT)(DG)(DT) (DC)

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Macromolecule #5: THYMIDINE-5'-TRIPHOSPHATE

MacromoleculeName: THYMIDINE-5'-TRIPHOSPHATE / type: ligand / ID: 5 / Number of copies: 1 / Formula: TTP
Molecular weightTheoretical: 482.168 Da
Chemical component information

ChemComp-TTP:
THYMIDINE-5'-TRIPHOSPHATE

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Macromolecule #6: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 6 / Number of copies: 3 / Formula: MG
Molecular weightTheoretical: 24.305 Da

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Macromolecule #7: 2',3'-DIDEOXYADENOSINE-5'-MONOPHOSPHATE

MacromoleculeName: 2',3'-DIDEOXYADENOSINE-5'-MONOPHOSPHATE / type: ligand / ID: 7 / Number of copies: 1 / Formula: 2DA
Molecular weightTheoretical: 315.222 Da
Chemical component information

ChemComp-2DA:
2',3'-DIDEOXYADENOSINE-5'-MONOPHOSPHATE

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Macromolecule #8: water

MacromoleculeName: water / type: ligand / ID: 8 / Number of copies: 2 / Formula: HOH
Molecular weightTheoretical: 18.015 Da
Chemical component information

ChemComp-HOH:
WATER

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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 2 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 complex of exonuclease deficient PolG, and RNA-DNA primer template, chain terminated with dideoxy ATP, with an incoming nucleotide (dTTP).

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Electron microscopy

MicroscopeTFS KRIOS
Specialist opticsEnergy filter - Slit width: 20 eV
SoftwareName: EPU
DetailsObjective aperture of 100 um was used
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Number grids imaged: 2 / Number real images: 12703 / Average electron dose: 70.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 1.8 µm / Nominal defocus min: 0.4 µm / Nominal magnification: 105000
Sample stageCooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

CTF correctionType: 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: 2.6 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 4.4) / Number images used: 1353452
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
FSC plot (resolution estimation)

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Atomic model buiding 1

RefinementSpace: REAL / Protocol: FLEXIBLE FIT
Output model

PDB-9pck:
Human DNA Polymerase Gamma-RNA-DNA Primer-Template Complex with Incoming ATP

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