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- PDB-8ucw: Complete DNA termination subcomplex 2 of Xenopus laevis DNA polym... -

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Basic information

Entry
Database: PDB / ID: 8ucw
TitleComplete DNA termination subcomplex 2 of Xenopus laevis DNA polymerase alpha-primase
Components
  • DNA polymerase alpha catalytic subunit
  • DNA template
  • RNA-DNA/DNA primer
KeywordsREPLICATION / TRANSFERASE/DNA/RNA / Primase / DNA polymerase / chimeric RNA-DNA primer / RNA/DNA hybrid / DNA replication / DNA synthesis / TRANSFERASE-DNA-RNA complex
Function / homology
Function and homology information


alpha DNA polymerase:primase complex / lagging strand elongation / DNA replication, synthesis of primer / mitotic DNA replication initiation / DNA strand elongation involved in DNA replication / leading strand elongation / DNA replication origin binding / DNA replication initiation / nuclear matrix / double-strand break repair via nonhomologous end joining ...alpha DNA polymerase:primase complex / lagging strand elongation / DNA replication, synthesis of primer / mitotic DNA replication initiation / DNA strand elongation involved in DNA replication / leading strand elongation / DNA replication origin binding / DNA replication initiation / nuclear matrix / double-strand break repair via nonhomologous end joining / nuclear envelope / single-stranded DNA binding / DNA replication / DNA-directed DNA polymerase / DNA-directed DNA polymerase activity / nucleotide binding / chromatin binding / chromatin / nucleolus / DNA binding / nucleoplasm / metal ion binding / nucleus
Similarity search - Function
DNA polymerase alpha catalytic subunit, N-terminal domain / DNA polymerase alpha, zinc finger domain superfamily / 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 family B, thumb domain / DNA polymerase family B signature. / DNA-directed DNA polymerase, family B, conserved site / DNA polymerase family B ...DNA polymerase alpha catalytic subunit, N-terminal domain / DNA polymerase alpha, zinc finger domain superfamily / 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 family B, thumb 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-directed DNA polymerase, family B, multifunctional 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
2'-DEOXYGUANOSINE-5'-TRIPHOSPHATE / DNA / DNA (> 10) / DNA/RNA hybrid / DNA/RNA hybrid (> 10) / DNA polymerase alpha catalytic subunit
Similarity search - Component
Biological speciesXenopus laevis (African clawed frog)
synthetic construct (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.64 Å
AuthorsMullins, E.A. / Chazin, W.C. / 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)CA92584 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
DepositionSep 27, 2023Deposition site: RCSB / Processing site: RCSB
Revision 1.0Oct 11, 2023Provider: repository / Type: Initial release
Revision 1.1Mar 20, 2024Group: Database references / Category: citation / citation_author
Revision 1.2Mar 27, 2024Group: Database references / Category: citation / citation_author
Item: _citation.pdbx_database_id_PubMed / _citation_author.identifier_ORCID
Revision 1.3May 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
C: DNA template
D: RNA-DNA/DNA primer
hetero molecules


Theoretical massNumber of molelcules
Total (without water)157,0196
Polymers156,4633
Non-polymers5563
Water0
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 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: DNA chain DNA template


Mass: 18203.668 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#3: DNA/RNA hybrid RNA-DNA/DNA primer


Mass: 9249.697 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#4: Chemical ChemComp-DGT / 2'-DEOXYGUANOSINE-5'-TRIPHOSPHATE / Deoxyguanosine triphosphate


Mass: 507.181 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C10H16N5O13P3 / Feature type: SUBJECT OF INVESTIGATION
#5: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Mg / Feature type: SUBJECT OF INVESTIGATION
Has ligand of interestY

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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-primase with a DNA termination substrateCOMPLEXHeterotetrameric protein complex with an RNA-DNA/DNA duplex#1-#30MULTIPLE SOURCES
2Polymerase alphaCOMPLEXHeterodimer consisting of POLA1 and POLA2#11RECOMBINANT
3PrimaseCOMPLEXHeterodimer consisting of PRIM1 and PRIM21RECOMBINANT
4DNA termination substrateCOMPLEXRNA-DNA/DNA duplex#2-#31NATURAL
Molecular weight
IDEntity assembly-IDValue (°)Experimental value
110.33 MDaNO
220.2 MDaNO
330.11 MDaNO
440.03 MDaNO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
12Xenopus laevis (African clawed frog)8355
23Xenopus laevis (African clawed frog)8355
34synthetic construct (others)32630
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 FIELDBright-field microscopy / Nominal magnification: 105000 X / Nominal defocus max: 2000 nm / Nominal defocus min: 800 nm / Cs: 2.7 mm
Image recordingElectron dose: 55.8 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 22893

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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
14PHENIXmodel refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 25216892
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.64 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 62414 / Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL
Atomic model building
IDPDB-ID 3D fitting-IDAccession codeInitial refinement model-IDSource nameType
18UCU

8ucu

18UCU1PDBexperimental model
28G9O

8g9o

18G9OPDBexperimental model
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0028562
ELECTRON MICROSCOPYf_angle_d0.46911876
ELECTRON MICROSCOPYf_dihedral_angle_d18.1221558
ELECTRON MICROSCOPYf_chiral_restr0.0411341
ELECTRON MICROSCOPYf_plane_restr0.0031294

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