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- EMDB-15309: Pol alpha - replisome complex containing Ctf4. Engaged on a DNA f... -

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Basic information

Entry
Database: EMDB / ID: EMD-15309
TitlePol alpha - replisome complex containing Ctf4. Engaged on a DNA fork containing a 60 nucleotide lagging strand.
Map dataPol alpha - replisome complex containing Ctf4. Unsharpened.
Sample
  • Complex: Replisome - pol alpha complex
KeywordsReplication / helicase / polymerase / pol alpha / priming
Function / homology
Function and homology information


Inhibition of replication initiation of damaged DNA by RB1/E2F1 / establishment of sister chromatid cohesion / H3-H4 histone complex chaperone activity / Unwinding of DNA / maintenance of DNA repeat elements / regulation of nuclear cell cycle DNA replication / DNA replication initiation / Cul8-RING ubiquitin ligase complex / replication fork arrest / RNA-templated DNA biosynthetic process ...Inhibition of replication initiation of damaged DNA by RB1/E2F1 / establishment of sister chromatid cohesion / H3-H4 histone complex chaperone activity / Unwinding of DNA / maintenance of DNA repeat elements / regulation of nuclear cell cycle DNA replication / DNA replication initiation / Cul8-RING ubiquitin ligase complex / replication fork arrest / RNA-templated DNA biosynthetic process / meiotic chromosome segregation / Processive synthesis on the lagging strand / DNA strand elongation involved in mitotic DNA replication / Removal of the Flap Intermediate / GINS complex / MCM core complex / Assembly of the pre-replicative complex / Switching of origins to a post-replicative state / MCM complex binding / mitotic DNA replication preinitiation complex assembly / nuclear DNA replication / Polymerase switching / premeiotic DNA replication / pre-replicative complex assembly involved in nuclear cell cycle DNA replication / anaphase-promoting complex binding / alpha DNA polymerase:primase complex / mitotic DNA replication / Activation of the pre-replicative complex / DNA replication checkpoint signaling / : / CMG complex / establishment of mitotic sister chromatid cohesion / nuclear pre-replicative complex / Activation of ATR in response to replication stress / telomere capping / DNA replication preinitiation complex / DNA replication, synthesis of primer / MCM complex / lagging strand elongation / replication fork protection complex / mitotic DNA replication checkpoint signaling / double-strand break repair via break-induced replication / single-stranded DNA helicase activity / mitotic DNA replication initiation / cellular response to osmotic stress / mitotic intra-S DNA damage checkpoint signaling / regulation of DNA-templated DNA replication initiation / silent mating-type cassette heterochromatin formation / mitotic sister chromatid cohesion / nuclear chromosome / DNA biosynthetic process / DNA synthesis involved in DNA repair / DNA strand elongation involved in DNA replication / leading strand elongation / mitotic G2 DNA damage checkpoint signaling / replication fork processing / nuclear replication fork / DNA replication origin binding / DNA replication initiation / Ub-specific processing proteases / subtelomeric heterochromatin formation / telomere maintenance / nuclear periphery / replication fork / meiotic cell cycle / helicase activity / transcription elongation by RNA polymerase II / DNA-templated DNA replication / Transferases; Transferring phosphorus-containing groups; Nucleotidyltransferases / nuclear envelope / heterochromatin formation / double-strand break repair / nucleosome assembly / mitotic cell cycle / single-stranded DNA binding / 4 iron, 4 sulfur cluster binding / DNA helicase / DNA-directed DNA polymerase / forked DNA-dependent helicase activity / single-stranded 3'-5' DNA helicase activity / four-way junction helicase activity / double-stranded DNA helicase activity / DNA-directed DNA polymerase activity / chromosome, telomeric region / DNA replication / protein stabilization / DNA repair / nucleotide binding / DNA damage response / chromatin binding / ATP hydrolysis activity / mitochondrion / DNA binding / zinc ion binding / nucleoplasm / ATP binding / metal ion binding / identical protein binding / nucleus / cytosol
Similarity search - Function
DNA replication checkpoint mediator, MRC1 domain / MRC1-like domain / Claspin / Chromosome segregation in meiosis protein 3 / TIPIN/Csm3/Swi3 / Replication Fork Protection Component Swi3 / : / DNA polymerase alpha subunit B, OB domain / Timeless, N-terminal / Timeless ...DNA replication checkpoint mediator, MRC1 domain / MRC1-like domain / Claspin / Chromosome segregation in meiosis protein 3 / TIPIN/Csm3/Swi3 / Replication Fork Protection Component Swi3 / : / DNA polymerase alpha subunit B, OB domain / Timeless, N-terminal / Timeless / Timeless protein / : / DNA polymerase alpha-binding protein Ctf4, C-terminal domain / DNA polymerase alpha, subunit B / Minichromosome loss protein Mcl1, middle region / Minichromosome loss protein, Mcl1, middle region / 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 / : / PSF3 N-terminal domain / CDC45 family / DNA replication complex GINS protein Psf2 / CDC45 / GINS complex, subunit Psf1 / GINS complex, subunit Psf3 / DNA replication complex GINS protein SLD5, C-terminal / GINS, helical bundle-like domain superfamily / GINS complex, subunit Psf3 superfamily / GINS complex protein Sld5, alpha-helical domain / : / DNA replication complex GINS protein SLD5 C-terminus / MCM3 winged helix domain / GINS complex subunit Sld5 / GINS subunit, domain A / GINS complex protein helical bundle domain / MCM4, winged helix domain / DNA polymerase alpha/delta/epsilon, subunit B / DNA polymerase alpha/epsilon subunit B / : / MCM5, C-terminal domain / DNA replication licensing factor Mcm5 / MCM3-like, winged helix domain / DNA replication licensing factor Mcm3 / Mini-chromosome maintenance complex protein 4 / DNA replication licensing factor Mcm6 / DNA replication licensing factor Mcm7 / Mcm6, C-terminal winged-helix domain / MCM6 C-terminal winged-helix domain / DNA replication licensing factor Mcm2 / Mini-chromosome maintenance protein 2 / Mini-chromosome maintenance, conserved site / MCM family signature. / MCM N-terminal domain / MCM N-terminal domain / MCM OB domain / MCM OB domain / Mini-chromosome maintenance protein / MCM, AAA-lid domain / MCM P-loop domain / MCM AAA-lid domain / MCM family domain profile. / minichromosome maintenance proteins / MCM domain / DNA polymerase family B, thumb domain / DNA polymerase family B signature. / DNA-directed DNA polymerase, family B, multifunctional domain / 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 / WD40-repeat-containing domain superfamily / Winged helix-like DNA-binding domain superfamily / WD40/YVTN repeat-like-containing domain superfamily / Nucleic acid-binding, OB-fold / ATPases associated with a variety of cellular activities / AAA+ ATPase domain / DNA/RNA polymerase superfamily / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
DNA primase small subunit / DNA polymerase alpha catalytic subunit A / DNA primase large subunit / DNA replication licensing factor MCM3 / Mediator of replication checkpoint protein 1 / DNA replication licensing factor MCM2 / Minichromosome maintenance protein 5 / DNA replication licensing factor MCM4 / DNA polymerase alpha subunit B / DNA replication licensing factor MCM7 ...DNA primase small subunit / DNA polymerase alpha catalytic subunit A / DNA primase large subunit / DNA replication licensing factor MCM3 / Mediator of replication checkpoint protein 1 / DNA replication licensing factor MCM2 / Minichromosome maintenance protein 5 / DNA replication licensing factor MCM4 / DNA polymerase alpha subunit B / DNA replication licensing factor MCM7 / DNA replication complex GINS protein PSF2 / DNA replication licensing factor MCM6 / Topoisomerase 1-associated factor 1 / DNA polymerase alpha-binding protein / DNA replication complex GINS protein SLD5 / Chromosome segregation in meiosis protein 3 / Cell division control protein 45 / DNA replication complex GINS protein PSF3 / DNA replication complex GINS protein PSF1
Similarity search - Component
Biological speciesSaccharomyces cerevisiae (brewer's yeast)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.64 Å
AuthorsJones ML / Yeeles JTP
Funding support United Kingdom, 1 items
OrganizationGrant numberCountry
Medical Research Council (MRC, United Kingdom)MC_UP_1201/12 United Kingdom
CitationJournal: Mol Cell / Year: 2023
Title: How Pol α-primase is targeted to replisomes to prime eukaryotic DNA replication.
Authors: Morgan L Jones / Valentina Aria / Yasemin Baris / Joseph T P Yeeles /
Abstract: During eukaryotic DNA replication, Pol α-primase generates primers at replication origins to start leading-strand synthesis and every few hundred nucleotides during discontinuous lagging-strand ...During eukaryotic DNA replication, Pol α-primase generates primers at replication origins to start leading-strand synthesis and every few hundred nucleotides during discontinuous lagging-strand replication. How Pol α-primase is targeted to replication forks to prime DNA synthesis is not fully understood. Here, by determining cryoelectron microscopy (cryo-EM) structures of budding yeast and human replisomes containing Pol α-primase, we reveal a conserved mechanism for the coordination of priming by the replisome. Pol α-primase binds directly to the leading edge of the CMG (CDC45-MCM-GINS) replicative helicase via a complex interaction network. The non-catalytic PRIM2/Pri2 subunit forms two interfaces with CMG that are critical for in vitro DNA replication and yeast cell growth. These interactions position the primase catalytic subunit PRIM1/Pri1 directly above the exit channel for lagging-strand template single-stranded DNA (ssDNA), revealing why priming occurs efficiently only on the lagging-strand template and elucidating a mechanism for Pol α-primase to overcome competition from RPA to initiate primer synthesis.
History
DepositionJul 4, 2022-
Header (metadata) releaseAug 2, 2023-
Map releaseAug 2, 2023-
UpdateAug 30, 2023-
Current statusAug 30, 2023Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_15309.png

Downloads & links

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Map

FileDownload / File: emd_15309.map.gz / Format: CCP4 / Size: 40.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationPol alpha - replisome complex containing Ctf4. Unsharpened.
Projections & slices

Image control

Size
Brightness
Contrast
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AxesZ (Sec.)Y (Row.)X (Col.)
2.27 Å/pix.
x 220 pix.
= 498.8 Å		2.27 Å/pix.
x 220 pix.
= 498.8 Å		2.27 Å/pix.
x 220 pix.
= 498.8 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

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Images are generated by Spider.

Voxel sizeX=Y=Z: 2.26727 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 1.43
Minimum - Maximum-3.500969 - 8.684388
Average (Standard dev.)0.0049348124 (±0.36013365)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions220220220
Spacing220220220
CellA=B=C: 498.8 Å
α=β=γ: 90.0 °

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Supplemental data

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Additional map: Locally filtered map

Fileemd_15309_additional_1.map
AnnotationLocally filtered map
Projections & Slices
AxesZYX

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Slices (1/2)
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Histogram

Histogram (log scale)

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Half map: Half map

Fileemd_15309_half_map_1.map
AnnotationHalf map
Projections & Slices
AxesZYX

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Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map

Fileemd_15309_half_map_2.map
AnnotationHalf map
Projections & Slices
AxesZYX

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Slices (1/2)
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Histogram

Histogram (log scale)

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Sample components

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Entire : Replisome - pol alpha complex

EntireName: Replisome - pol alpha complex
Components
  • Complex: Replisome - pol alpha complex

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Supramolecule #1: Replisome - pol alpha complex

SupramoleculeName: Replisome - pol alpha complex / type: complex / ID: 1 / Parent: 0
Details: S. cerevisiae pol alpha bound to the core replisome engaged with a fork DNA substrate containing a 60 nucleotide lagging strand.
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)

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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

BufferpH: 7.6
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: 40.184 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 3.5 µm / Nominal defocus min: 1.5 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: OTHER
Final reconstructionResolution.type: BY AUTHOR / Resolution: 4.64 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 24127
Initial angle assignmentType: ANGULAR RECONSTITUTION
Final angle assignmentType: ANGULAR RECONSTITUTION
FSC plot (resolution estimation)

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