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- EMDB-29751: Cryo-EM structure of the Guide loop Engagement Complex (IV) of Hu... -

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Entry
Database: EMDB / ID: EMD-29751
TitleCryo-EM structure of the Guide loop Engagement Complex (IV) of Human Mitochondrial DNA Polymerase Gamma
Map dataCryo-EM structure of the Guide loop Engagement Complex (IV) of Human Mitochondrial DNA Polymerase Gamma
Sample
  • Complex: Cryo-EM structure of the Guide Loop Engagement Complex (IV) of Human Mitochondrial DNA Polymerase Gamma
    • Protein or peptide: DNA polymerase subunit gamma-1
    • Protein or peptide: DNA polymerase subunit gamma-2, mitochondrial
    • RNA: Mismatched RNA primer
    • DNA: Template DNA
KeywordsMitochondrial DNA Polymerase / DNA Proofreading / Guide loop Engagement / REPLICATION-DNA-RNA 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 polymerase subunit gamma-1 / DNA polymerase subunit gamma-2
Similarity search - Component
Biological speciesHomo sapiens (human) / synthetic RNA (others) / synthetic construct (others)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.61 Å
AuthorsNayak AR / Buchel G / Herbine KH / Sarfallah A / Sokolova VO / Zamudio-Ochoa A / Temiakov D
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: 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, 2023-
Header (metadata) releaseJan 10, 2024-
Map releaseJan 10, 2024-
UpdateJan 10, 2024-
Current statusJan 10, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_29751.png

Downloads & links

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Map

FileDownload / File: emd_29751.map.gz / Format: CCP4 / Size: 115.9 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationCryo-EM structure of the Guide loop Engagement Complex (IV) of Human Mitochondrial DNA Polymerase Gamma
Voxel sizeX=Y=Z: 0.8256 Å
Density
Contour LevelBy AUTHOR: 0.05
Minimum - Maximum-0.2863069 - 0.5564348
Average (Standard dev.)0.00037410477 (±0.012439954)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderZYX
Origin000
Dimensions312312312
Spacing312312312
CellA=B=C: 257.58722 Å
α=β=γ: 90.0 °

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Supplemental data

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Additional map: Sharpened map

Fileemd_29751_additional_1.map
AnnotationSharpened map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: Masked half map B

Fileemd_29751_additional_2.map
AnnotationMasked half map B
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: Masked half map A

Fileemd_29751_additional_3.map
AnnotationMasked half map A
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Raw unfiltered half map A

Fileemd_29751_half_map_1.map
AnnotationRaw unfiltered half map A
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Raw unfiltered half map B

Fileemd_29751_half_map_2.map
AnnotationRaw unfiltered half map B
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Cryo-EM structure of the Guide Loop Engagement Complex (IV) of Hu...

EntireName: Cryo-EM structure of the Guide Loop Engagement Complex (IV) of Human Mitochondrial DNA Polymerase Gamma
Components
  • Complex: Cryo-EM structure of the Guide Loop Engagement Complex (IV) of Human Mitochondrial DNA Polymerase Gamma
    • Protein or peptide: DNA polymerase subunit gamma-1
    • Protein or peptide: DNA polymerase subunit gamma-2, mitochondrial
    • RNA: Mismatched RNA primer
    • DNA: Template DNA

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Supramolecule #1: Cryo-EM structure of the Guide Loop Engagement Complex (IV) of Hu...

SupramoleculeName: Cryo-EM structure of the Guide Loop Engagement Complex (IV) of Human Mitochondrial DNA Polymerase Gamma
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Details: Human mitochondrial DNA polymerase PolG (exonuclease deficient D198A/E200A variant) assembled on an RNA-DNA scaffold in the presence of GTP
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: 139.628672 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 IPEERALVFA VAVCLAEGTC 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: Mismatched RNA primer

MacromoleculeName: Mismatched RNA primer / type: rna / ID: 3 / Number of copies: 1
Source (natural)Organism: synthetic RNA (others)
Molecular weightTheoretical: 7.857758 KDa
SequenceString:
GAAGACAGUC UGCGGCGCGC GGGG

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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: 9.176879 KDa
SequenceString:
(DG)(DG)(DT)(DA)(DG)(DA)(DT)(DC)(DC)(DC) (DG)(DC)(DG)(DC)(DG)(DC)(DC)(DG)(DC)(DA) (DG)(DA)(DC)(DT)(DG)(DT)(DC)(DT)(DT) (DC)

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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 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Atmosphere: AIR
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV
Details1:1 complex of Exo- PolG and RNA-DNA scaffold in the presence of 0.1 mM dGTP

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 2.0 µm / Nominal defocus min: 0.8 µm / Nominal magnification: 105000
Specialist opticsEnergy filter - Slit width: 20 eV
Sample stageCooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Number grids imaged: 2 / Number real images: 12937 / Average electron dose: 60.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 14667792
Startup modelType of model: NONE / Details: Ab-initio reconstruction in cryoSPARC
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final 3D classificationNumber classes: 4 / Avg.num./class: 426672 / Software - Name: cryoSPARC (ver. 3.2)
Details: The final classification into four classes, resulted in a 3Dclass with 426672 particles that yielded the G-loop engagement (IV) complex.
Final angle assignmentType: MAXIMUM LIKELIHOOD
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 2.61 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 3.2) / Number images used: 426672
FSC plot (resolution estimation)

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Atomic model buiding 1

RefinementSpace: REAL / Protocol: FLEXIBLE FIT
Output model

PDB-8g5o:
Cryo-EM structure of the Guide loop Engagement Complex (IV) of Human Mitochondrial DNA Polymerase Gamma

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