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- PDB-8g9f: Complete auto-inhibitory complex of Xenopus laevis DNA polymerase... -

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Basic information

Entry
Database: PDB / ID: 8g9f
TitleComplete auto-inhibitory complex of Xenopus laevis DNA polymerase alpha-primase
Components
  • (DNA polymerase alpha ...) x 2
  • DNA primase
  • DNA primase large subunit
KeywordsREPLICATION / TRANSFERASE / Primase / DNA polymerase / chimeric RNA-DNA primer / RNA/DNA hybrid / DNA replication / DNA synthesis
Function / homology
Function and homology information


alpha DNA polymerase:primase complex / DNA primase activity / primosome complex / DNA replication, synthesis of primer / lagging strand elongation / mitotic DNA replication initiation / DNA strand elongation involved in DNA replication / leading strand elongation / DNA replication initiation / DNA-directed RNA polymerase complex ...alpha DNA polymerase:primase complex / DNA primase activity / primosome complex / DNA replication, synthesis of primer / lagging strand elongation / mitotic DNA replication initiation / DNA strand elongation involved in DNA replication / leading strand elongation / DNA replication initiation / DNA-directed RNA polymerase complex / double-strand break repair via nonhomologous end joining / nuclear matrix / Transferases; Transferring phosphorus-containing groups; Nucleotidyltransferases / nuclear envelope / 4 iron, 4 sulfur cluster binding / DNA replication / DNA-directed DNA polymerase / DNA-directed DNA polymerase activity / nucleotide binding / chromatin binding / chromatin / nucleolus / DNA binding / nucleoplasm / nucleus / metal ion binding
Similarity search - Function
DNA polymerase alpha, subunit B, N-terminal domain superfamily / : / DNA polymerase alpha subunit B, OB domain / DNA polymerase alpha subunit B N-terminal / DNA polymerase alpha, subunit B, N-terminal / DNA polymerase alpha, subunit B / DNA primase, small subunit, eukaryotic/archaeal / DNA primase, large subunit, eukaryotic / DNA primase, small subunit / DNA primase small subunit ...DNA polymerase alpha, subunit B, N-terminal domain superfamily / : / DNA polymerase alpha subunit B, OB domain / DNA polymerase alpha subunit B N-terminal / DNA polymerase alpha, subunit B, N-terminal / DNA polymerase alpha, subunit B / DNA primase, small subunit, eukaryotic/archaeal / DNA primase, large subunit, eukaryotic / DNA primase, small subunit / DNA primase small subunit / DNA primase large subunit, eukaryotic/archaeal / DNA polymerase alpha catalytic subunit, N-terminal domain / DNA polymerase alpha, zinc finger domain superfamily / Eukaryotic and archaeal DNA primase, large subunit / DNA Polymerase alpha zinc finger / DNA polymerase alpha subunit p180 N terminal / Zinc finger, DNA-directed DNA polymerase, family B, alpha / DNA polymerase alpha catalytic subunit, catalytic domain / DNA polymerase alpha/delta/epsilon, subunit B / DNA polymerase alpha/epsilon subunit B / DNA polymerase family B, thumb domain / DNA-directed DNA polymerase, family B, multifunctional domain / DNA polymerase family B signature. / DNA-directed DNA polymerase, family B, conserved site / DNA polymerase family B / DNA polymerase family B, exonuclease domain / DNA-directed DNA polymerase, family B, exonuclease domain / DNA polymerase, palm domain superfamily / DNA polymerase type-B family / DNA-directed DNA polymerase, family B / Ribonuclease H superfamily / Ribonuclease H-like superfamily / DNA/RNA polymerase superfamily
Similarity search - Domain/homology
IRON/SULFUR CLUSTER / DNA primase large subunit / DNA polymerase alpha subunit B / DNA primase / DNA polymerase alpha catalytic subunit
Similarity search - Component
Biological speciesXenopus laevis (African clawed frog)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.2 Å
AuthorsMullins, E.A. / Chazin, W.J. / Eichman, B.F.
Funding support United States, 3items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM136401 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM118089 United States
National Institutes of Health/National Cancer Institute (NIH/NCI)PO1CA92584 United States
Citation
Journal: Nat Struct Mol Biol / Year: 2024
Title: A mechanistic model of primer synthesis from catalytic structures of DNA polymerase α-primase.
Authors: Elwood A Mullins / Lauren E Salay / Clarissa L Durie / Noah P Bradley / Jane E Jackman / Melanie D Ohi / Walter J Chazin / Brandt F Eichman /
Abstract: The mechanism by which polymerase α-primase (polα-primase) synthesizes chimeric RNA-DNA primers of defined length and composition, necessary for replication fidelity and genome stability, is ...The mechanism by which polymerase α-primase (polα-primase) synthesizes chimeric RNA-DNA primers of defined length and composition, necessary for replication fidelity and genome stability, is unknown. Here, we report cryo-EM structures of Xenopus laevis polα-primase in complex with primed templates representing various stages of DNA synthesis. Our data show how interaction of the primase regulatory subunit with the primer 5' end facilitates handoff of the primer to polα and increases polα processivity, thereby regulating both RNA and DNA composition. The structures detail how flexibility within the heterotetramer enables synthesis across two active sites and provide evidence that termination of DNA synthesis is facilitated by reduction of polα and primase affinities for the varied conformations along the chimeric primer-template duplex. Together, these findings elucidate a critical catalytic step in replication initiation and provide a comprehensive model for primer synthesis by polα-primase.
#1: Journal: bioRxiv / Year: 2023
Title: A mechanistic model of primer synthesis from catalytic structures of DNA polymerase α-primase.
Authors: Elwood A Mullins / Lauren E Salay / Clarissa L Durie / Noah P Bradley / Jane E Jackman / Melanie D Ohi / Walter J Chazin / Brandt F Eichman
Abstract: The mechanism by which polymerase α-primase (polα-primase) synthesizes chimeric RNA-DNA primers of defined length and composition, necessary for replication fidelity and genome stability, is ...The mechanism by which polymerase α-primase (polα-primase) synthesizes chimeric RNA-DNA primers of defined length and composition, necessary for replication fidelity and genome stability, is unknown. Here, we report cryo-EM structures of polα-primase in complex with primed templates representing various stages of DNA synthesis. Our data show how interaction of the primase regulatory subunit with the primer 5'-end facilitates handoff of the primer to polα and increases polα processivity, thereby regulating both RNA and DNA composition. The structures detail how flexibility within the heterotetramer enables synthesis across two active sites and provide evidence that termination of DNA synthesis is facilitated by reduction of polα and primase affinities for the varied conformations along the chimeric primer/template duplex. Together, these findings elucidate a critical catalytic step in replication initiation and provide a comprehensive model for primer synthesis by polα-primase.
History
DepositionFeb 21, 2023Deposition site: RCSB / Processing site: RCSB
Revision 1.0Apr 12, 2023Provider: repository / Type: Initial release
Revision 1.1Oct 11, 2023Group: Data collection / Database references / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / citation_author / em_3d_fitting_list
Item: _em_3d_fitting_list.initial_refinement_model_id
Revision 1.2Mar 20, 2024Group: Database references / Category: citation / citation_author
Revision 1.3Mar 27, 2024Group: Database references / Category: citation / citation_author
Item: _citation.pdbx_database_id_PubMed / _citation_author.identifier_ORCID
Revision 1.4Apr 24, 2024Group: Refinement description / Category: em_3d_fitting / em_3d_fitting_list
Revision 1.5May 29, 2024Group: Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first / _citation.page_last

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: DNA polymerase alpha catalytic subunit
B: DNA polymerase alpha subunit B
C: DNA primase large subunit
D: DNA primase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)305,7828
Polymers305,2344
Non-polymers5484
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration, electron microscopy
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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DNA polymerase alpha ... , 2 types, 2 molecules AB

#1: Protein DNA polymerase alpha catalytic subunit / DNA polymerase alpha catalytic subunit p180


Mass: 129009.414 Da / Num. of mol.: 1 / Fragment: UNP residues 335-1458
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Xenopus laevis (African clawed frog) / Gene: pola1, pola / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: Q9DE46, DNA-directed DNA polymerase
#2: Protein DNA polymerase alpha subunit B


Mass: 67194.219 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Xenopus laevis (African clawed frog) / Gene: pola2.L / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: Q6DCZ1

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Protein , 2 types, 2 molecules CD

#3: Protein DNA primase large subunit


Mass: 59673.844 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Xenopus laevis (African clawed frog) / Gene: prim2.S / Production host: Escherichia coli (E. coli) / References: UniProt: A0A1L8G3G3
#4: Protein DNA primase


Mass: 49356.387 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Xenopus laevis (African clawed frog) / Gene: prim1.S / Production host: Escherichia coli (E. coli) / References: UniProt: Q800A4

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Non-polymers , 2 types, 4 molecules

#5: Chemical ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: Zn
#6: Chemical ChemComp-SF4 / IRON/SULFUR CLUSTER


Mass: 351.640 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Fe4S4

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Details

Has ligand of interestN

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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
IDNameTypeDetailsEntity IDParent-IDSource
1Polymerase alpha-primaseCOMPLEXHeterotetrameric protein complex#1-#40MULTIPLE SOURCES
2Polymerase alphaCOMPLEXHeterodimer consisting of POLA1 and POLA2#1-#21RECOMBINANT
3PrimaseCOMPLEXHeterodimer consisting of PRIM1 and PRIM2#3-#41RECOMBINANT
Molecular weight
IDEntity assembly-IDValue (°)Experimental value
110.3 MDaNO
220.2 MDaNO
310.11 MDaNO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
12Xenopus laevis (African clawed frog)8355
23Xenopus laevis (African clawed frog)8355
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
12Spodoptera frugiperda (fall armyworm)7108
23Escherichia coli (E. coli)562
Buffer solutionpH: 7.5
SpecimenConc.: 0.9 mg/ml / Embedding 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 magnification: 105000 X / Nominal defocus max: 2000 nm / Nominal defocus min: 800 nm / Cs: 2.7 mm
Image recordingElectron dose: 54.8 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 17820

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Processing

EM software
IDNameCategory
1cryoSPARCparticle selection
2Topazparticle selection
3SerialEMimage acquisition
5MotionCorr2CTF correction
8Cootmodel fitting
10cryoSPARCinitial Euler assignment
11RELIONfinal Euler assignment
13RELION3D reconstruction
20PHENIXmodel refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 23007566
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.2 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 47277 / Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL
Atomic model buildingPDB-ID: 8G99

8g99


Accession code: 8G99 / Source name: PDB / Type: experimental model
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00319203
ELECTRON MICROSCOPYf_angle_d0.49125963
ELECTRON MICROSCOPYf_dihedral_angle_d4.1242539
ELECTRON MICROSCOPYf_chiral_restr0.042851
ELECTRON MICROSCOPYf_plane_restr0.0043335

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