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- PDB-8udl: Human Mitochondrial DNA Polymerase Gamma Binary Complex -

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Basic information

Entry
Database: PDB / ID: 8udl
TitleHuman Mitochondrial DNA Polymerase Gamma Binary Complex
Components
  • DNA (5'-D(P*AP*AP*AP*AP*CP*GP*AP*CP*GP*GP*CP*CP*AP*GP*TP*GP*CP*CP*AP*TP*AP*C)-3')
  • DNA (5'-D(P*AP*GP*GP*TP*AP*TP*GP*GP*CP*AP*CP*TP*GP*GP*CP*CP*GP*TP*CP*GP*TP*TP*TP*T)-3')
  • DNA polymerase subunit gamma-1
  • DNA polymerase subunit gamma-2, mitochondrial
KeywordsTRANSFERASE/DNA / Mitochondrial / DNA Polymerase / TRANSFERASE / TRANSFERASE-DNA complex
Function / homology
Function and homology information


gamma DNA polymerase complex / mitochondrial DNA replication / positive regulation of DNA-directed DNA polymerase activity / single-stranded DNA 3'-5' DNA exonuclease activity / Hydrolases; Acting on ester bonds; Exodeoxyribonucleases producing 5'-phosphomonoesters / DNA replication proofreading / 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 / single-stranded DNA 3'-5' DNA exonuclease activity / Hydrolases; Acting on ester bonds; Exodeoxyribonucleases producing 5'-phosphomonoesters / DNA replication proofreading / 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 / 3'-5' exonuclease activity / base-excision repair, gap-filling / Transcriptional activation of mitochondrial biogenesis / base-excision repair / 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 / 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: 2.37 Å
AuthorsPark, J. / Yin, Y.W.
Funding support United States, 2items
OrganizationGrant numberCountry
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)R01AI134611 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01GM145925 United States
CitationJournal: Sci Adv / Year: 2024
Title: An interaction network in the polymerase active site is a prerequisite for Watson-Crick base pairing in Pol γ.
Authors: Joon Park / Geoffrey K Herrmann / Arkanil Roy / Christie K Shumate / G Andrés Cisneros / Y Whitney Yin /
Abstract: The replication accuracy of DNA polymerase gamma (Pol γ) is essential for mitochondrial genome integrity. Mutation of human Pol γ arginine-853 has been linked to neurological diseases. Although not ...The replication accuracy of DNA polymerase gamma (Pol γ) is essential for mitochondrial genome integrity. Mutation of human Pol γ arginine-853 has been linked to neurological diseases. Although not a catalytic residue, Pol γ arginine-853 mutants are void of polymerase activity. To identify the structural basis for the disease, we determined a crystal structure of the Pol γ mutant ternary complex with correct incoming nucleotide 2'-deoxycytidine 5'-triphosphate (dCTP). Opposite to the wild type that undergoes open-to-closed conformational changes when bound to a correct nucleotide that is essential for forming a catalytically competent active site, the mutant complex failed to undergo the conformational change, and the dCTP did not base pair with its Watson-Crick complementary templating residue. Our studies revealed that arginine-853 coordinates an interaction network that aligns the 3'-end of primer and dCTP with the catalytic residues. Disruption of the network precludes the formation of Watson-Crick base pairing and closing of the active site, resulting in an inactive polymerase.
History
DepositionSep 28, 2023Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jun 5, 2024Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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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: DNA (5'-D(P*AP*AP*AP*AP*CP*GP*AP*CP*GP*GP*CP*CP*AP*GP*TP*GP*CP*CP*AP*TP*AP*C)-3')
T: DNA (5'-D(P*AP*GP*GP*TP*AP*TP*GP*GP*CP*AP*CP*TP*GP*GP*CP*CP*GP*TP*CP*GP*TP*TP*TP*T)-3')


Theoretical massNumber of molelcules
Total (without water)263,8605
Polymers263,8605
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#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
Production host: Insect cell expression vector pTIE1 (others)
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
Production host: Insect cell expression vector pTIE1 (others)
References: UniProt: Q9UHN1, DNA-directed DNA polymerase
#3: DNA chain DNA (5'-D(P*AP*AP*AP*AP*CP*GP*AP*CP*GP*GP*CP*CP*AP*GP*TP*GP*CP*CP*AP*TP*AP*C)-3')


Mass: 6739.384 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#4: DNA chain DNA (5'-D(P*AP*GP*GP*TP*AP*TP*GP*GP*CP*AP*CP*TP*GP*GP*CP*CP*GP*TP*CP*GP*TP*TP*TP*T)-3')


Mass: 7407.761 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)

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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

Component
IDNameTypeEntity IDParent-IDSource
1Human Mitochondrial DNA Polymerase Gamma Binary ComplexCOMPLEXall0MULTIPLE SOURCES
2DNA polymerase subunit gamma-1,DNA polymerase subunit gamma-2COMPLEX#1-#21RECOMBINANT
3DNACOMPLEX#3-#41SYNTHETIC
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Insect cell expression vector pTIE1 (others)
Buffer solutionpH: 7.5
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 2500 nm / Nominal defocus min: 1500 nm
Image recordingElectron dose: 50 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k)

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Processing

CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 2.37 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 2027209 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00315728
ELECTRON MICROSCOPYf_angle_d0.73621512
ELECTRON MICROSCOPYf_dihedral_angle_d15.3355900
ELECTRON MICROSCOPYf_chiral_restr0.0442323
ELECTRON MICROSCOPYf_plane_restr0.0112602

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