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- EMDB-42984: Active conformation of DNA polymerase gamma bound to DNA -

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Basic information

Entry
Database: EMDB / ID: EMD-42984
TitleActive conformation of DNA polymerase gamma bound to DNA
Map data
Sample
  • Complex: DNA polymerase gamma complex in an active conformation
    • Protein or peptide: DNA polymerase subunit gamma-1
    • Protein or peptide: DNA polymerase subunit gamma-2, mitochondrial
    • DNA: primer DNA
    • DNA: Template DNA
  • Ligand: MAGNESIUM ION
  • Ligand: 2',3'-DIDEOXY-THYMIDINE-5'-TRIPHOSPHATE
KeywordsDNA BINDING PROTEIN / DNA polymerase / mitochondrial DNA replication / DNA polymerase gamma / DNA BINDING PROTEIN-DNA 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 / base-excision repair / Transcriptional activation of mitochondrial biogenesis / DNA-templated DNA replication / double-stranded DNA binding / protease binding / in utero embryonic development / 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 / Anticodon-binding domain superfamily / DNA-directed DNA polymerase, family A, conserved site ...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 / 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)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.0 Å
AuthorsRiccio AA / Krahn JM / Bouvette J / Borgnia JM / Copeland WC
Funding support United States, 2 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of Environmental Health Sciences (NIH/NIEHS)Z01 ES065078 United States
National Institutes of Health/National Institute of Environmental Health Sciences (NIH/NIEHS)Z01 ES065080 United States
CitationJournal: Nucleic Acids Res / Year: 2024
Title: Coordinated DNA polymerization by Polγ and the region of LonP1 regulated proteolysis.
Authors: Amanda A Riccio / Asia J Brannon / Juno M Krahn / Jonathan Bouvette / Jason G Williams / Mario J Borgnia / William C Copeland /
Abstract: The replicative mitochondrial DNA polymerase, Polγ, and its protein regulation are essential for the integrity of the mitochondrial genome. The intricacies of Polγ regulation and its interactions ...The replicative mitochondrial DNA polymerase, Polγ, and its protein regulation are essential for the integrity of the mitochondrial genome. The intricacies of Polγ regulation and its interactions with regulatory proteins, which are essential for fine-tuning polymerase function, remain poorly understood. Misregulation of the Polγ heterotrimer, consisting of (i) PolG, the polymerase catalytic subunit and (ii) PolG2, the accessory subunit, ultimately results in mitochondrial diseases. Here, we used single particle cryo-electron microscopy to resolve the structure of PolG in its apoprotein state and we captured Polγ at three intermediates within the catalytic cycle: DNA bound, engaged, and an active polymerization state. Chemical crosslinking mass spectrometry, and site-directed mutagenesis uncovered the region of LonP1 engagement of PolG, which promoted proteolysis and regulation of PolG protein levels. PolG2 clinical variants, which disrupted a stable Polγ complex, led to enhanced LonP1-mediated PolG degradation. Overall, this insight into Polγ aids in an understanding of mitochondrial DNA replication and characterizes how machinery of the replication fork may be targeted for proteolytic degradation when improperly functioning.
History
DepositionNov 30, 2023-
Header (metadata) releaseJul 10, 2024-
Map releaseJul 10, 2024-
UpdateJul 31, 2024-
Current statusJul 31, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_42984.png

Downloads & links

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Map

FileDownload / File: emd_42984.map.gz / Format: CCP4 / Size: 107.2 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.83 Å/pix.
x 304 pix.
= 253.232 Å		0.83 Å/pix.
x 304 pix.
= 253.232 Å		0.83 Å/pix.
x 304 pix.
= 253.232 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.833 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.0305
Minimum - Maximum-0.03128004 - 2.1134596
Average (Standard dev.)0.002068063 (±0.03285171)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions304304304
Spacing304304304
CellA=B=C: 253.232 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #2

Fileemd_42984_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_42984_half_map_2.map
Projections & Slices
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Histogram (log scale)

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Sample components

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Entire : DNA polymerase gamma complex in an active conformation

EntireName: DNA polymerase gamma complex in an active conformation
Components
  • Complex: DNA polymerase gamma complex in an active conformation
    • Protein or peptide: DNA polymerase subunit gamma-1
    • Protein or peptide: DNA polymerase subunit gamma-2, mitochondrial
    • DNA: primer DNA
    • DNA: Template DNA
  • Ligand: MAGNESIUM ION
  • Ligand: 2',3'-DIDEOXY-THYMIDINE-5'-TRIPHOSPHATE

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Supramolecule #1: DNA polymerase gamma complex in an active conformation

SupramoleculeName: DNA polymerase gamma complex in an active conformation
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#4
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 504 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
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 138.723438 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: MGGSHHHHHH GSRFMVSSSV PASDPSDGQR RRQQQQQQQQ QQQQQPQQPQ VLSSEGGQLR HNPLDIQMLS RGLHEQIFGQ GGEMPGEAA VRRSVEHLQK HGLWGQPAVP LPDVELRLPP LYGDNLDQHF RLLAQKQSLP YLEAANLLLQ AQLPPKPPAW A WAEGWTRY ...String:
MGGSHHHHHH GSRFMVSSSV PASDPSDGQR RRQQQQQQQQ QQQQQPQQPQ VLSSEGGQLR HNPLDIQMLS RGLHEQIFGQ GGEMPGEAA VRRSVEHLQK HGLWGQPAVP LPDVELRLPP LYGDNLDQHF RLLAQKQSLP YLEAANLLLQ AQLPPKPPAW A WAEGWTRY GPEGEAVPVA IPEERALVFA VAVCLAEGTC PTLAVAISPS AWYSWCSQRL VEERYSWTSQ LSPADLIPLE VP TGASSPT QRDWQEQLVV GHNVSFDRAH IREQYLIQGS RMRFLDTMSM HMAISGLSSF QRSLWIAAKQ GKHKVQPPTK QGQ KSQRKA RRGPAISSWD WLDISSVNSL AEVHRLYVGG PPLEKEPREL FVKGTMKDIR ENFQDLMQYC AQDVWATHEV FQQQ LPLFL ERCPHPVTLA GMLEMGVSYL PVNQNWERYL AEAQGTYEEL QREMKKSLMD LANDACQLLS GERYKEDPWL WDLEW DLQE FKQKKAKKVK KEPATASKLP IEGAGAPGDP MDQEDLGPCS EEEEFQQDVM ARACLQKLKG TTELLPKRPQ HLPGHP GWY RKLCPRLDDP AWTPGPSLLS LQMRVTPKLM ALTWDGFPLH YSERHGWGYL VPGRRDNLAK LPTGTTLESA GVVCPYR AI ESLYRKHCLE QGKQQLMPQE AGLAEEFLLT DNSAIWQTVE ELDYLEVEAE AKMENLRAAV PGQPLALTAR GGPKDTQP S YHHGNGPYND VDIPGCWFFK LPHKDGNSCN VGSPFAKDFL PKMEDGTLQA GPGGASGPRA LEINKMISFW RNAHKRISS QMVVWLPRSA LPRAVIRHPD YDEEGLYGAI LPQVVTAGTI TRRAVEPTWL TASNARPDRV GSELKAMVQA PPGYTLVGAD VDSQELWIA AVLGDAHFAG MHGCTAFGWM TLQGRKSRGT DLHSKTATTV GISREHAKIF NYGRIYGAGQ PFAERLLMQF N HRLTQQEA AEKAQQMYAA TKGLRWYRLS DEGEWLVREL NLPVDRTEGG WISLQDLRKV QRETARKSQW KKWEVVAERA WK GGTESEM FNKLESIATS DIPRTPVLGC CISRALEPSA VQEEFMTSRV NWVVQSSAVD YLHLMLVAMK WLFEEFAIDG RFC ISIHDE VRYLVREEDR YRAALALQIT NLLTRCMFAY KLGLNDLPQS VAFFSAVDID RCLRKEVTMD CKTPSNPTGM ERRY GIPQG 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
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 53.787281 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MASRGSHHHH HHGADAGQPE LLTERSSPKG GHVKSHAELE GNGEHPEAPG SGEGSEALLE ICQRRHFLSG SKQQLSRDSL LSGCHPGFG PLGVELRKNL AAEWWTSVVV FREQVFPVDA LHHKPGPLLP GDSAFRLVSA ETLREILQDK ELSKEQLVAF L ENVLKTSG ...String:
MASRGSHHHH HHGADAGQPE LLTERSSPKG GHVKSHAELE GNGEHPEAPG SGEGSEALLE ICQRRHFLSG SKQQLSRDSL LSGCHPGFG PLGVELRKNL AAEWWTSVVV FREQVFPVDA LHHKPGPLLP GDSAFRLVSA ETLREILQDK ELSKEQLVAF L ENVLKTSG KLRENLLHGA LEHYVNCLDL VNKRLPYGLA QIGVCFHPVF DTKQIRNGVK SIGEKTEASL VWFTPPRTSN QW LDFWLRH RLQWWRKFAM SPSNFSSSDC QDEEGRKGNK LYYNFPWGKE LIETLWNLGD HELLHMYPGN VSKLHGRDGR KNV VPCVLS VNGDLDRGML AYLYDSFQLT ENSFTRKKNL HRKVLKLHPC LAPIKVALDV GRGPTLELRQ VCQGLFNELL ENGI SVWPG YLETMQSSLE QLYSKYDEMS ILFTVLVTET TLENGLIHLR SRDTTMKEMM HISKLKDFLI KYISSAKNV

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: Homo sapiens (human)
Molecular weightTheoretical: 15.780146 KDa
SequenceString:
(DT)(DG)(DG)(DG)(DT)(DT)(DT)(DT)(DT)(DA) (DT)(DG)(DT)(DA)(DC)(DT)(DA)(DC)(DA)(DG) (DG)(DT)(DG)(DG)(DT)(DC)(DA)(DA)(DG) (DT)(DA)(DT)(DT)(DT)(DA)(DT)(DG)(DG)(DT) (DA) (DC)(DC)(DG)(DT)(DA)(DC)(DA)(DA) (DT)(DA)(2DT)

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Macromolecule #4: Template DNA

MacromoleculeName: Template DNA / type: dna / ID: 4 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 24.477756 KDa
SequenceString: (DA)(DT)(DT)(DT)(DC)(DG)(DT)(DA)(DC)(DA) (DT)(DT)(DA)(DC)(DT)(DG)(DC)(DC)(DA)(DG) (DC)(DC)(DA)(DC)(DC)(DA)(DT)(DG)(DA) (DA)(DT)(DA)(DT)(DT)(DG)(DT)(DA)(DC)(DG) (DG) (DT)(DA)(DC)(DC)(DA)(DT) ...String:
(DA)(DT)(DT)(DT)(DC)(DG)(DT)(DA)(DC)(DA) (DT)(DT)(DA)(DC)(DT)(DG)(DC)(DC)(DA)(DG) (DC)(DC)(DA)(DC)(DC)(DA)(DT)(DG)(DA) (DA)(DT)(DA)(DT)(DT)(DG)(DT)(DA)(DC)(DG) (DG) (DT)(DA)(DC)(DC)(DA)(DT)(DA)(DA) (DA)(DT)(DA)(DC)(DT)(DT)(DG)(DA)(DC)(DC) (DA)(DC) (DC)(DT)(DG)(DT)(DA)(DG)(DT) (DA)(DC)(DA)(DT)(DA)(DA)(DA)(DA)(DA)(DC) (DC)(DC)(DA)

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Macromolecule #5: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 5 / Number of copies: 1 / Formula: MG
Molecular weightTheoretical: 24.305 Da

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Macromolecule #6: 2',3'-DIDEOXY-THYMIDINE-5'-TRIPHOSPHATE

MacromoleculeName: 2',3'-DIDEOXY-THYMIDINE-5'-TRIPHOSPHATE / type: ligand / ID: 6 / Number of copies: 1 / Formula: D3T
Molecular weightTheoretical: 466.169 Da
Chemical component information

ChemComp-23T:
2',3'-DIDEOXY-THYMIDINE-5'-TRIPHOSPHATE

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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

Concentration1.2 mg/mL
BufferpH: 8
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeTFS KRIOS
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Average electron dose: 60.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.5 µm / Nominal defocus min: 1.0 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: OTHER
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.0 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 208863
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
FSC plot (resolution estimation)

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