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- EMDB-17795: Cryo-EM structure of human DNA polymerase alpha-primase in pre-in... -

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Basic information

Entry
Database: EMDB / ID: EMD-17795
TitleCryo-EM structure of human DNA polymerase alpha-primase in pre-initiation stage 1
Map data
Sample
  • Complex: DNA polymerase alpha-primase with bound nucleotide
KeywordsDNA polymerase / complex / DNA BINDING PROTEIN
Function / homology
Function and homology information


positive regulation of DNA primase activity / DNA primase AEP / ribonucleotide binding / DNA replication initiation / DNA/RNA hybrid binding / Telomere C-strand synthesis initiation / Inhibition of replication initiation of damaged DNA by RB1/E2F1 / regulation of type I interferon production / Polymerase switching / alpha DNA polymerase:primase complex ...positive regulation of DNA primase activity / DNA primase AEP / ribonucleotide binding / DNA replication initiation / DNA/RNA hybrid binding / Telomere C-strand synthesis initiation / Inhibition of replication initiation of damaged DNA by RB1/E2F1 / regulation of type I interferon production / Polymerase switching / alpha DNA polymerase:primase complex / Processive synthesis on the lagging strand / DNA primase activity / Removal of the Flap Intermediate / Polymerase switching on the C-strand of the telomere / primosome complex / DNA replication, synthesis of primer / lagging strand elongation / mitotic DNA replication initiation / DNA strand elongation involved in DNA replication / DNA synthesis involved in DNA repair / leading strand elongation / G1/S-Specific Transcription / DNA replication origin binding / DNA replication initiation / Activation of the pre-replicative complex / Defective pyroptosis / double-strand break repair via nonhomologous end joining / nuclear matrix / protein import into nucleus / nuclear envelope / single-stranded DNA binding / 4 iron, 4 sulfur cluster binding / DNA replication / DNA-directed DNA polymerase / DNA-directed DNA polymerase activity / DNA repair / nucleotide binding / chromatin binding / chromatin / nucleolus / protein kinase binding / magnesium ion binding / DNA binding / zinc ion binding / nucleoplasm / membrane / nucleus / metal ion binding / cytosol
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
DNA polymerase alpha catalytic subunit / DNA primase small subunit / DNA primase large subunit / DNA polymerase alpha subunit B
Similarity search - Component
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.07 Å
AuthorsYin Z / Pellegrini L
Funding support United Kingdom, 1 items
OrganizationGrant numberCountry
Wellcome Trust221892/Z/20/Z United Kingdom
CitationJournal: FEBS J / Year: 2024
Title: CryoEM insights into RNA primer synthesis by the human primosome.
Authors: Zhan Yin / Mairi L Kilkenny / De-Sheng Ker / Luca Pellegrini /
Abstract: Eukaryotic DNA replication depends on the primosome - a complex of DNA polymerase alpha (Pol α) and primase - to initiate DNA synthesis by polymerisation of an RNA-DNA primer. Primer synthesis ...Eukaryotic DNA replication depends on the primosome - a complex of DNA polymerase alpha (Pol α) and primase - to initiate DNA synthesis by polymerisation of an RNA-DNA primer. Primer synthesis requires the tight coordination of primase and polymerase activities. Recent cryo-electron microscopy (cryoEM) analyses have elucidated the extensive conformational transitions required for RNA primer handover between primase and Pol α and primer elongation by Pol α. Because of the intrinsic flexibility of the primosome, however, structural information about the initiation of RNA primer synthesis is still lacking. Here, we capture cryoEM snapshots of the priming reaction to reveal the conformational trajectory of the human primosome that brings DNA primase subunits 1 and 2 (PRIM1 and PRIM2, respectively) together, poised for RNA synthesis. Furthermore, we provide experimental evidence for the continuous association of primase subunit PRIM2 with the RNA primer during primer synthesis, and for how both initiation and termination of RNA primer polymerisation are licenced by specific rearrangements of DNA polymerase alpha catalytic subunit (POLA1), the polymerase subunit of Pol α. Our findings fill a critical gap in our understanding of the conformational changes that underpin the synthesis of the RNA primer by the primosome. Together with existing evidence, they provide a complete description of the structural dynamics of the human primosome during DNA replication initiation.
History
DepositionJul 6, 2023-
Header (metadata) releaseFeb 21, 2024-
Map releaseFeb 21, 2024-
UpdateMay 1, 2024-
Current statusMay 1, 2024Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_17795.png

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Map

FileDownload / File: emd_17795.map.gz / Format: CCP4 / Size: 209.3 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

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AxesZ (Sec.)Y (Row.)X (Col.)
1.07 Å/pix.
x 380 pix.
= 405.08 Å		1.07 Å/pix.
x 380 pix.
= 405.08 Å		1.07 Å/pix.
x 380 pix.
= 405.08 Å

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Images are generated by Spider.

Voxel sizeX=Y=Z: 1.066 Å
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Histogram (log scale)
Contour LevelBy AUTHOR: 0.147
Minimum - Maximum-0.34298632 - 1.6732529
Average (Standard dev.)0.0004403696 (±0.018731037)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions380380380
Spacing380380380
CellA=B=C: 405.08 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_17795_msk_1.map
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Half map: #1

Fileemd_17795_half_map_1.map
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Half map: #2

Fileemd_17795_half_map_2.map
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Sample components

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Entire : DNA polymerase alpha-primase with bound nucleotide

EntireName: DNA polymerase alpha-primase with bound nucleotide
Components
  • Complex: DNA polymerase alpha-primase with bound nucleotide

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Supramolecule #1: DNA polymerase alpha-primase with bound nucleotide

SupramoleculeName: DNA polymerase alpha-primase with bound nucleotide / type: complex / ID: 1 / Parent: 0
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 287.5 KDa

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration0.29 mg/mL
BufferpH: 7.4
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 47.78 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.6 µm / Nominal defocus min: 1.0 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: NONE
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.07 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 185009
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC / Details: ab-initio models in
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC / Details: Non-uniform refinement
FSC plot (resolution estimation)

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