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- PDB-8v6h: DNA initiation complex (configuration 2) of Xenopus laevis DNA po... -

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Basic information

Entry
Database: PDB / ID: 8v6h
TitleDNA initiation complex (configuration 2) of Xenopus laevis DNA polymerase alpha-primase
Components
  • (DNA polymerase alpha ...DNA polymerase) x 2
  • DNA primase large subunitPrimase
  • DNA primasePrimase
  • DNA template
  • RNA primerPrimer (molecular biology)
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 / DNA primase activity / lagging strand elongation / DNA replication, synthesis of primer / mitotic DNA replication initiation / DNA strand elongation involved in DNA replication / leading strand elongation / DNA replication initiation / DNA-directed RNA polymerase complex / nuclear matrix ...alpha DNA polymerase:primase complex / DNA primase activity / lagging strand elongation / DNA replication, synthesis of primer / mitotic DNA replication initiation / DNA strand elongation involved in DNA replication / leading strand elongation / DNA replication initiation / DNA-directed RNA polymerase complex / nuclear matrix / double-strand break repair via nonhomologous end joining / 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 / metal ion binding / nucleus
Similarity search - Function
DNA polymerase alpha, subunit B, N-terminal domain superfamily / 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, subunit B, N-terminal domain superfamily / 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 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 / IRON/SULFUR CLUSTER / DNA / DNA (> 10) / RNA / 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)
synthetic construct (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 11.11 Å
AuthorsMullins, E.A. / Durie, C.L. / Ohi, M.D. / 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)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: To Be Published
Title: A mechanistic model of primer synthesis from catalytic structures of DNA polymerase alpha-primase
Authors: Mullins, E.A. / Salay, L.E. / Durie, C.L. / Bradley, N.P. / Jackman, J.E. / Ohi, M.D. / Chazin, W.J. / Eichman, B.F.
History
DepositionDec 1, 2023Deposition site: RCSB / Processing site: RCSB
Revision 1.0Dec 20, 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

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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
E: DNA template
F: RNA primer
hetero molecules


Theoretical massNumber of molelcules
Total (without water)324,79613
Polymers323,6936
Non-polymers1,1047
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, not applicable
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 / DNA polymerase


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 / Primase


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 / 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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DNA chain / RNA chain , 2 types, 2 molecules EF

#5: DNA chain DNA template


Mass: 15458.913 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#6: RNA chain RNA primer / Primer (molecular biology)


Mass: 2999.727 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)

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Non-polymers , 4 types, 7 molecules

#7: Chemical ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: Zn
#8: Chemical ChemComp-DGT / 2'-DEOXYGUANOSINE-5'-TRIPHOSPHATE / Deoxyguanosine triphosphate


Mass: 507.181 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C10H16N5O13P3
#9: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Mg
#10: Chemical ChemComp-SF4 / IRON/SULFUR CLUSTER / 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-primase with a DNA initiation substrateCOMPLEXHeterotetrameric protein complex with an RNA/DNA duplex#1-#60MULTIPLE SOURCES
2Polymerase alphaCOMPLEXHeterodimer consisting of POLA1 and POLA2#1-#21RECOMBINANT
3PrimaseCOMPLEXHeterodimer consisting of PRIM1 and PRIM2#3-#41RECOMBINANT
4DNA initiation substrateCOMPLEXRNA/DNA duplex#5-#61NATURAL
Molecular weight
IDEntity assembly-IDValue (°)Experimental value
110.32 MDaNO
210.2 MDaNO
310.11 MDaNO
440.02 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: Talos Arctica / Image courtesy: FEI Company
MicroscopyModel: FEI TALOS ARCTICA
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 45000 X / Nominal defocus max: 2500 nm / Nominal defocus min: 1500 nm / Cs: 2.7 mm
Image recordingElectron dose: 50.7 e/Å2 / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of grids imaged: 4 / Num. of real images: 12845

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Processing

EM software
IDNameCategory
1cryoSPARCparticle selection
2Topazparticle selection
3Leginonimage acquisition
5cryoSPARCCTF correction
8UCSF ChimeraXmodel fitting
10cryoSPARCinitial Euler assignment
11cryoSPARCfinal Euler assignment
13cryoSPARC3D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 11200341
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 11.11 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 28068 / Symmetry type: POINT
Atomic model building
IDProtocolSpace
1RIGID BODY FITREAL
2
3
4
5
6
Atomic model building
IDPDB-ID 3D fitting-IDAccession codeInitial refinement model-IDSource nameType
18G9F

8g9f

18G9F1PDBexperimental model
28G9L

8g9l

18G9L2PDBexperimental model
32
43
54
65
76

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