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- PDB-7uy8: Tetrahymena Polymerase alpha-Primase -

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Basic information

Entry
Database: PDB / ID: 7uy8
TitleTetrahymena Polymerase alpha-Primase
Components
  • DNA polymerase
  • DNA polymerase alpha subunit B
  • DNA primase
  • Eukaryotic-type DNA primase, large subunit
KeywordsREPLICATION / CST / polymerase / primase
Function / homology
Function and homology information


alpha DNA polymerase:primase complex / lagging strand elongation / mitotic DNA replication initiation / DNA replication, synthesis of primer / leading strand elongation / DNA replication origin binding / DNA replication initiation / DNA-directed RNA polymerase complex / Transferases; Transferring phosphorus-containing groups; Nucleotidyltransferases / DNA-directed RNA polymerase activity ...alpha DNA polymerase:primase complex / lagging strand elongation / mitotic DNA replication initiation / DNA replication, synthesis of primer / leading strand elongation / DNA replication origin binding / DNA replication initiation / DNA-directed RNA polymerase complex / Transferases; Transferring phosphorus-containing groups; Nucleotidyltransferases / DNA-directed RNA polymerase activity / single-stranded DNA binding / 4 iron, 4 sulfur cluster binding / DNA-directed DNA polymerase / DNA-directed DNA polymerase activity / nucleotide binding / chromatin binding / DNA binding / metal ion binding
Similarity search - Function
: / DNA polymerase alpha subunit B, OB domain / 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 / Eukaryotic and archaeal DNA primase, large subunit C-terminal domain / DNA polymerase alpha catalytic subunit, catalytic domain ...: / DNA polymerase alpha subunit B, OB domain / 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 / Eukaryotic and archaeal DNA primase, large subunit C-terminal domain / 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-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 / DNA/RNA polymerase superfamily
Similarity search - Domain/homology
DNA polymerase alpha subunit B / DNA polymerase / Eukaryotic-type DNA primase, large subunit / DNA primase
Similarity search - Component
Biological speciesTetrahymena thermophila (eukaryote)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4.5 Å
AuthorsHe, Y. / Song, H. / Chan, H. / Wang, Y. / Liu, B. / Susac, L. / Zhou, Z.H. / Feigon, J.
Funding support United States, 3items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R35GM131901 United States
National Science Foundation (NSF, United States)MCB2016540 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01GM071940 United States
CitationJournal: Nature / Year: 2022
Title: Structure of Tetrahymena telomerase-bound CST with polymerase α-primase.
Authors: Yao He / He Song / Henry Chan / Baocheng Liu / Yaqiang Wang / Lukas Sušac / Z Hong Zhou / Juli Feigon /
Abstract: Telomeres are the physical ends of linear chromosomes. They are composed of short repeating sequences (such as TTGGGG in the G-strand for Tetrahymena thermophila) of double-stranded DNA with a single- ...Telomeres are the physical ends of linear chromosomes. They are composed of short repeating sequences (such as TTGGGG in the G-strand for Tetrahymena thermophila) of double-stranded DNA with a single-strand 3' overhang of the G-strand and, in humans, the six shelterin proteins: TPP1, POT1, TRF1, TRF2, RAP1 and TIN2. TPP1 and POT1 associate with the 3' overhang, with POT1 binding the G-strand and TPP1 (in complex with TIN2) recruiting telomerase via interaction with telomerase reverse transcriptase (TERT). The telomere DNA ends are replicated and maintained by telomerase, for the G-strand, and subsequently DNA polymerase α-primase (PolαPrim), for the C-strand. PolαPrim activity is stimulated by the heterotrimeric complex CTC1-STN1-TEN1 (CST), but the structural basis of the recruitment of PolαPrim and CST to telomere ends remains unknown. Here we report cryo-electron microscopy (cryo-EM) structures of Tetrahymena CST in the context of the telomerase holoenzyme, in both the absence and the presence of PolαPrim, and of PolαPrim alone. Tetrahymena Ctc1 binds telomerase subunit p50, a TPP1 orthologue, on a flexible Ctc1 binding motif revealed by cryo-EM and NMR spectroscopy. The PolαPrim polymerase subunit POLA1 binds Ctc1 and Stn1, and its interface with Ctc1 forms an entry port for G-strand DNA to the POLA1 active site. We thus provide a snapshot of four key components that are required for telomeric DNA synthesis in a single active complex-telomerase-core ribonucleoprotein, p50, CST and PolαPrim-that provides insights into the recruitment of CST and PolαPrim and the handoff between G-strand and C-strand synthesis.
History
DepositionMay 6, 2022Deposition site: RCSB / Processing site: RCSB
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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
B: DNA polymerase alpha subunit B
A: DNA polymerase
D: Eukaryotic-type DNA primase, large subunit
C: DNA primase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)342,3865
Polymers342,3204
Non-polymers651
Water00
1


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

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Components

#1: Protein DNA polymerase alpha subunit B


Mass: 68773.758 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Tetrahymena thermophila (eukaryote) / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: I7MAE1
#2: Protein DNA polymerase


Mass: 161986.984 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Tetrahymena thermophila (eukaryote) / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: Q23AJ0, DNA-directed DNA polymerase
#3: Protein Eukaryotic-type DNA primase, large subunit


Mass: 64386.645 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Tetrahymena thermophila (eukaryote) / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: Q246C7
#4: Protein DNA primase


Mass: 47172.855 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Tetrahymena thermophila (eukaryote) / Production host: Spodoptera frugiperda (fall armyworm)
References: UniProt: Q24HY6, Transferases; Transferring phosphorus-containing groups; Nucleotidyltransferases
#5: Chemical ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Zn / Feature type: SUBJECT OF INVESTIGATION
Has ligand of interestY
Has protein modificationN

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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: Tetrahymena PolaPrim / Type: COMPLEX / Entity ID: #1-#4 / Source: RECOMBINANT
Source (natural)Organism: Tetrahymena thermophila (eukaryote)
Source (recombinant)Organism: Spodoptera frugiperda (fall armyworm)
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: 4000 nm / Nominal defocus min: 800 nm
Image recordingElectron dose: 55 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

SoftwareName: PHENIX / Version: 1.19.2_4158: / Classification: refinement
EM softwareName: PHENIX / Category: model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 4.5 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 264498 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0039719
ELECTRON MICROSCOPYf_angle_d0.63613079
ELECTRON MICROSCOPYf_dihedral_angle_d14.5543725
ELECTRON MICROSCOPYf_chiral_restr0.0461473
ELECTRON MICROSCOPYf_plane_restr0.0041659

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