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- PDB-8d0k: Human CST-DNA polymerase alpha/primase preinitiation complex boun... -

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Basic information

Entry
Database: PDB / ID: 8d0k
TitleHuman CST-DNA polymerase alpha/primase preinitiation complex bound to 4xTEL-foldback template - PRIM2C advanced PIC
Components
  • (CST complex subunit ...) x 3
  • (DNA polymerase alpha ...) x 2
  • DNA (5'-D(P*TP*AP*GP*GP*GP*TP*TP*AP*GP*GP*GP*TP*TP*AP*GP*GP*GP*TP*TP*A)-3')
  • DNA primase large subunit
  • DNA primase small subunit
KeywordsREPLICATION/DNA / telomere / C-strand / complex / 4xTEL-foldback DNA template / PRIM2C / REPLICATION-DNA complex
Function / homology
Function and homology information


CST complex / DNA primase AEP / ribonucleotide binding / telomerase inhibitor activity / DNA replication initiation / telomere maintenance via telomere lengthening / Telomere C-strand synthesis initiation / DNA/RNA hybrid binding / Inhibition of replication initiation of damaged DNA by RB1/E2F1 / regulation of type I interferon production ...CST complex / DNA primase AEP / ribonucleotide binding / telomerase inhibitor activity / DNA replication initiation / telomere maintenance via telomere lengthening / Telomere C-strand synthesis initiation / DNA/RNA hybrid binding / Inhibition of replication initiation of damaged DNA by RB1/E2F1 / regulation of type I interferon production / alpha DNA polymerase:primase complex / Polymerase switching / Processive synthesis on the lagging strand / single-stranded telomeric DNA binding / G-rich strand telomeric DNA binding / lagging strand elongation / Removal of the Flap Intermediate / telomere capping / intermediate filament cytoskeleton / Polymerase switching on the C-strand of the telomere / mitotic DNA replication initiation / DNA replication, synthesis of primer / bone marrow development / DNA strand elongation involved in DNA replication / DNA synthesis involved in DNA repair / telomeric DNA binding / hematopoietic stem cell proliferation / leading strand elongation / G1/S-Specific Transcription / negative regulation of telomere maintenance via telomerase / DNA replication origin binding / replicative senescence / DNA replication initiation / Activation of the pre-replicative complex / spleen development / regulation of G2/M transition of mitotic cell cycle / telomere maintenance / thymus development / positive regulation of DNA replication / Defective pyroptosis / double-strand break repair via nonhomologous end joining / multicellular organism growth / nuclear matrix / fibrillar center / positive regulation of fibroblast proliferation / protein import into nucleus / DNA-directed RNA polymerase activity / nuclear envelope / single-stranded DNA binding / 4 iron, 4 sulfur cluster binding / DNA-directed DNA polymerase / DNA-directed DNA polymerase activity / chromosome, telomeric region / DNA replication / ciliary basal body / DNA repair / nucleotide binding / intracellular membrane-bounded organelle / DNA damage response / chromatin binding / protein kinase binding / chromatin / nucleolus / magnesium ion binding / DNA binding / zinc ion binding / nucleoplasm / metal ion binding / nucleus / membrane / cytosol
Similarity search - Function
CST complex subunit Ten1, animal and plant type / CST complex subunit CTC1 / CST complex subunit CTC1-like / CST, telomere maintenance, complex subunit CTC1 / Telomere-capping, CST complex subunit / CST complex subunit Stn1 / Stn1, C-terminal / CST complex subunit Stn1, wHTH1 motif superfamily / CST, complex subunit STN1, C terminal / DNA polymerase alpha, subunit B, N-terminal domain superfamily ...CST complex subunit Ten1, animal and plant type / CST complex subunit CTC1 / CST complex subunit CTC1-like / CST, telomere maintenance, complex subunit CTC1 / Telomere-capping, CST complex subunit / CST complex subunit Stn1 / Stn1, C-terminal / CST complex subunit Stn1, wHTH1 motif superfamily / CST, complex subunit STN1, C terminal / DNA polymerase alpha, subunit B, N-terminal domain superfamily / Replication factor A protein-like / : / 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 C-terminal domain / 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 / OB-fold nucleic acid binding domain, AA-tRNA synthetase-type / OB-fold nucleic acid binding domain / DNA polymerase family B, thumb domain / DNA-directed DNA polymerase, family B, multifunctional domain / DNA-directed DNA polymerase, family B, conserved site / DNA polymerase family B signature. / 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 / Winged helix DNA-binding domain superfamily / Winged helix-like DNA-binding domain superfamily / Nucleic acid-binding, OB-fold / DNA/RNA polymerase superfamily
Similarity search - Domain/homology
: / DNA / DNA (> 10) / DNA polymerase alpha catalytic subunit / DNA primase small subunit / DNA primase large subunit / DNA polymerase alpha subunit B / CST complex subunit CTC1 / CST complex subunit TEN1 / CST complex subunit STN1
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4.27 Å
AuthorsHe, Q. / Lin, X. / Chavez, B.L. / Agrawal, S. / Lusk, B.L. / Lim, C.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R00GM131023 United States
CitationJournal: Nature / Year: 2022
Title: Structures of the human CST-Polα-primase complex bound to telomere templates.
Authors: Qixiang He / Xiuhua Lin / Bianca L Chavez / Sourav Agrawal / Benjamin L Lusk / Ci Ji Lim /
Abstract: The mammalian DNA polymerase-α-primase (Polα-primase) complex is essential for DNA metabolism, providing the de novo RNA-DNA primer for several DNA replication pathways such as lagging-strand ...The mammalian DNA polymerase-α-primase (Polα-primase) complex is essential for DNA metabolism, providing the de novo RNA-DNA primer for several DNA replication pathways such as lagging-strand synthesis and telomere C-strand fill-in. The physical mechanism underlying how Polα-primase, alone or in partnership with accessory proteins, performs its complicated multistep primer synthesis function is unknown. Here we show that CST, a single-stranded DNA-binding accessory protein complex for Polα-primase, physically organizes the enzyme for efficient primer synthesis. Cryogenic electron microscopy structures of the CST-Polα-primase preinitiation complex (PIC) bound to various types of telomere overhang reveal that template-bound CST partitions the DNA and RNA catalytic centres of Polα-primase into two separate domains and effectively arranges them in RNA-DNA synthesis order. The architecture of the PIC provides a single solution for the multiple structural requirements for the synthesis of RNA-DNA primers by Polα-primase. Several insights into the template-binding specificity of CST, template requirement for assembly of the CST-Polα-primase PIC and activation are also revealed in this study.
History
DepositionMay 26, 2022Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jun 22, 2022Provider: repository / Type: Initial release
Revision 1.1Aug 17, 2022Group: Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_ASTM / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year / _citation_author.identifier_ORCID / _citation_author.name
Revision 1.2Sep 7, 2022Group: Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation_author.identifier_ORCID
Revision 1.3Feb 14, 2024Group: Data collection / Category: chem_comp_atom / chem_comp_bond

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: CST complex subunit CTC1
B: CST complex subunit STN1
C: CST complex subunit TEN1
D: DNA primase small subunit
E: DNA primase large subunit
F: DNA polymerase alpha catalytic subunit
G: DNA polymerase alpha subunit B
H: DNA (5'-D(P*TP*AP*GP*GP*GP*TP*TP*AP*GP*GP*GP*TP*TP*AP*GP*GP*GP*TP*TP*A)-3')


Theoretical massNumber of molelcules
Total (without water)568,7998
Polymers568,7998
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, Cryo-EM reconstruction
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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CST complex subunit ... , 3 types, 3 molecules ABC

#1: Protein CST complex subunit CTC1


Mass: 138030.250 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: Q2NKJ3
#2: Protein CST complex subunit STN1 / Oligonucleotide/oligosaccharide-binding fold-containing protein 1 / Suppressor of cdc thirteen homolog


Mass: 43001.824 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: STN1, OBFC1 / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: Q9H668
#3: Protein CST complex subunit TEN1 / Protein telomeric pathways with STN1 homolog / Telomere length regulation protein TEN1 homolog


Mass: 17285.604 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: TEN1, C17orf106 / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: Q86WV5

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Protein , 2 types, 2 molecules DE

#4: Protein DNA primase small subunit / DNA primase 49 kDa subunit / p49


Mass: 51356.379 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: PRIM1 / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: P49642, DNA primase AEP
#5: Protein DNA primase large subunit / DNA primase 58 kDa subunit / p58


Mass: 60266.293 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: PRIM2, PRIM2A / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: P49643

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

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


Mass: 172796.859 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: POLA1, POLA / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: P09884, DNA-directed DNA polymerase
#7: Protein DNA polymerase alpha subunit B / DNA polymerase alpha 70 kDa subunit


Mass: 67390.898 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: POLA2 / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: Q14181

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DNA chain , 1 types, 1 molecules H

#8: DNA chain DNA (5'-D(P*TP*AP*GP*GP*GP*TP*TP*AP*GP*GP*GP*TP*TP*AP*GP*GP*GP*TP*TP*A)-3')


Mass: 18670.920 Da / Num. of mol.: 1 / Source method: obtained synthetically
Details: Residue 1-35 forms a double-stranded DNA hairpin stem at the 5' end of the single-stranded DNA telomeric template.
Source: (synth.) Homo sapiens (human) / References: GenBank: 1894914083

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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

ComponentName: Human CST-DNA polymerase alpha/primase advanced preinitiation complex bound to 4xTEL-foldback DNA template
Type: COMPLEX
Details: Fold-back double-stranded DNA region of the DNA template is not modeled due to structural flexibility.
Entity ID: all / Source: RECOMBINANT
Molecular weightValue: 0.54 MDa / Experimental value: YES
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Trichoplusia ni (cabbage looper)
Buffer solutionpH: 7.5
Details: CHAPSO is only added just before sample vitrification.
Buffer component
IDConc.NameBuffer-ID
150 mMHEPES-Na1
2150 mMNaCl1
32 mMMgCl21
41 mMTCEP1
54 mMCHAPSO1
SpecimenConc.: 5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 95 % / Chamber temperature: 277.15 K

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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: SPOT SCAN
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 2800 nm / Nominal defocus min: 700 nm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 50 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 7794

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Processing

SoftwareName: PHENIX / Version: 1.20.1_4487: / Classification: refinement
EM software
IDNameCategory
2SerialEMimage acquisition
4CTFFINDCTF correction
7Cootmodel fitting
9PHENIXmodel refinement
12RELIONclassification
13cryoSPARC3D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 4.27 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 32550 / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT / Space: REAL / Target criteria: cross coefficient
Details: Alphafold models were used to dock into the map before coot adjustments and phenix refinement.
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00231773
ELECTRON MICROSCOPYf_angle_d0.4943122
ELECTRON MICROSCOPYf_dihedral_angle_d8.6064351
ELECTRON MICROSCOPYf_chiral_restr0.0394774
ELECTRON MICROSCOPYf_plane_restr0.0035479

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