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- EMDB-29873: Complete DNA elongation subcomplex of Xenopus laevis DNA polymera... -

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Basic information

Entry
Database: EMDB / ID: EMD-29873
TitleComplete DNA elongation subcomplex of Xenopus laevis DNA polymerase alpha-primase
Map dataComplete DNA elongation subcomplex of Xenopus laevis DNA polymerase alpha-primase
Sample
  • Complex: Polymerase alpha-primase with a DNA elongation substrate
    • Complex: Polymerase alpha
      • Protein or peptide: DNA polymerase alpha catalytic subunit
    • Complex: Primase
      • Protein or peptide: DNA primase large subunit
    • Complex: DNA elongation substrate
      • DNA: DNA template
      • Other: RNA-DNA primer
  • Ligand: 2'-DEOXYGUANOSINE-5'-TRIPHOSPHATE
  • Ligand: MAGNESIUM ION
  • Ligand: IRON/SULFUR CLUSTER
KeywordsPrimase / DNA polymerase / chimeric RNA-DNA primer / RNA/DNA hybrid / DNA replication / DNA synthesis / REPLICATION / TRANSFERASE-DNA-RNA complex
Function / homology
Function and homology information


alpha DNA polymerase:primase complex / lagging strand elongation / mitotic DNA replication initiation / DNA replication, synthesis of primer / DNA strand elongation involved in DNA replication / leading strand elongation / DNA replication origin binding / DNA replication initiation / double-strand break repair via nonhomologous end joining / nuclear matrix ...alpha DNA polymerase:primase complex / lagging strand elongation / mitotic DNA replication initiation / DNA replication, synthesis of primer / DNA strand elongation involved in DNA replication / leading strand elongation / DNA replication origin binding / DNA replication initiation / double-strand break repair via nonhomologous end joining / nuclear matrix / nuclear envelope / single-stranded DNA binding / 4 iron, 4 sulfur cluster binding / DNA-directed DNA polymerase / DNA-directed DNA polymerase activity / DNA replication / nucleotide binding / chromatin binding / chromatin / nucleolus / DNA binding / zinc ion binding / nucleoplasm / metal ion binding / nucleus
Similarity search - Function
DNA primase, large subunit, eukaryotic / 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 family B, thumb domain ...DNA primase, large subunit, eukaryotic / 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 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 primase large subunit / DNA polymerase alpha catalytic subunit
Similarity search - Component
Biological speciesXenopus laevis (African clawed frog) / synthetic construct (others)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.4 Å
AuthorsMullins EA / Durie CL / Ohi MD / Chazin WJ / Eichman BF
Funding support United States, 3 items
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)PO1CA92584 United States
CitationJournal: bioRxiv / Year: 2023
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 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.
History
DepositionFeb 21, 2023-
Header (metadata) releaseApr 12, 2023-
Map releaseApr 12, 2023-
UpdateMay 29, 2024-
Current statusMay 29, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_29873.png

Downloads & links

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Map

FileDownload / File: emd_29873.map.gz / Format: CCP4 / Size: 52.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationComplete DNA elongation subcomplex of Xenopus laevis DNA polymerase alpha-primase
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.37 Å/pix.
x 240 pix.
= 327.6 Å		1.37 Å/pix.
x 240 pix.
= 327.6 Å		1.37 Å/pix.
x 240 pix.
= 327.6 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.365 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.015
Minimum - Maximum-0.019745981 - 0.068589136
Average (Standard dev.)-0.000005490286 (±0.0017880506)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions240240240
Spacing240240240
CellA=B=C: 327.6 Å
α=β=γ: 90.0 °

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Supplemental data

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Additional map: Complete DNA elongation subcomplex of Xenopus laevis DNA...

Fileemd_29873_additional_1.map
AnnotationComplete DNA elongation subcomplex of Xenopus laevis DNA polymerase alpha-primase (locally sharpened)
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Complete DNA elongation subcomplex of Xenopus laevis DNA...

Fileemd_29873_half_map_1.map
AnnotationComplete DNA elongation subcomplex of Xenopus laevis DNA polymerase alpha-primase (half 1)
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Complete DNA elongation subcomplex of Xenopus laevis DNA...

Fileemd_29873_half_map_2.map
AnnotationComplete DNA elongation subcomplex of Xenopus laevis DNA polymerase alpha-primase (half 2)
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Polymerase alpha-primase with a DNA elongation substrate

EntireName: Polymerase alpha-primase with a DNA elongation substrate
Components
  • Complex: Polymerase alpha-primase with a DNA elongation substrate
    • Complex: Polymerase alpha
      • Protein or peptide: DNA polymerase alpha catalytic subunit
    • Complex: Primase
      • Protein or peptide: DNA primase large subunit
    • Complex: DNA elongation substrate
      • DNA: DNA template
      • Other: RNA-DNA primer
  • Ligand: 2'-DEOXYGUANOSINE-5'-TRIPHOSPHATE
  • Ligand: MAGNESIUM ION
  • Ligand: IRON/SULFUR CLUSTER

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Supramolecule #1: Polymerase alpha-primase with a DNA elongation substrate

SupramoleculeName: Polymerase alpha-primase with a DNA elongation substrate
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#4
Details: Heterotetrameric protein complex with an RNA-DNA/DNA duplex
Molecular weightTheoretical: 110 KDa

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Supramolecule #2: Polymerase alpha

SupramoleculeName: Polymerase alpha / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1 / Details: Heterodimer consisting of POLA1 and POLA2
Source (natural)Organism: Xenopus laevis (African clawed frog)

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Supramolecule #3: Primase

SupramoleculeName: Primase / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #2 / Details: Heterodimer consisting of PRIM1 and PRIM2
Source (natural)Organism: Xenopus laevis (African clawed frog)

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Supramolecule #4: DNA elongation substrate

SupramoleculeName: DNA elongation substrate / type: complex / ID: 4 / Parent: 1 / Macromolecule list: #3-#4 / Details: RNA-DNA/DNA duplex
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 20 KDa

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Macromolecule #1: DNA polymerase alpha catalytic subunit

MacromoleculeName: DNA polymerase alpha catalytic subunit / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO / EC number: DNA-directed DNA polymerase
Source (natural)Organism: Xenopus laevis (African clawed frog)
Molecular weightTheoretical: 129.009414 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: SNAADGSQVF RFYWLDAYED QYSQPGVVYL FGKVWIESAD AYVSCCVSVK NIERTVYLLP RENRVQLSTG KDTGAPVSMM HVYQEFNEA VAEKYKIMKF KSKKVDKDYA FEIPDVPASS EYLEVRYSAD SPQLPQDLKG ETFSHVFGTN TSSLELFLLS R KIKGPSWL ...String:
SNAADGSQVF RFYWLDAYED QYSQPGVVYL FGKVWIESAD AYVSCCVSVK NIERTVYLLP RENRVQLSTG KDTGAPVSMM HVYQEFNEA VAEKYKIMKF KSKKVDKDYA FEIPDVPASS EYLEVRYSAD SPQLPQDLKG ETFSHVFGTN TSSLELFLLS R KIKGPSWL EIKSPQLSSQ PMSWCKVEAV VTRPDQVSVV KDLAPPPVVV LSLSMKTVQN AKTHQNEIVA IAALVHHTFP LD KAPPQPP FQTHFCVLSK LNDCIFPYDY NEAVKQKNAN IEIALTERTL LGFFLAKIHK IDPDVIVGHD IYGFDLEVLL QRI NSCKVP FWSKIGRLRR SVMPKLGGRS GFAERNAACG RIICDIEISA KELIRCKSYH LSELVHQILK AERVVIPPEN IRNA YNDSV HLLYMLENTW IDAKFILQIM CELNVLPLAL QITNIAGNVM SRTLMGGRSE RNEYLLLHAF TENNFIVPDK PVFKK MQQT TVEDNDDMGT DQNKNKSRKK AAYAGGLVLE PKVGFYDKFI LLLDFNSLYP SIIQEYNICF TTVHREAPST QKGEDQ DEI PELPHSDLEM GILPREIRKL VERRRHVKQL MKQPDLNPDL YLQYDIRQKA LKLTANSMYG CLGFSYSRFY AKPLAAL VT HQGREILLHT KEMVQKMNLE VIYGDTDSIM INTNCNNLEE VFKLGNRVKS EINKSYKLLE IDIDGIFKSL LLLKKKKY A ALTVEPTGDG KYVTKQELKG LDIVRRDWCE LAKQAGNYVI SQILSDQPRD SIVENIQKKL TEIGENVTNG TVPITQYEI NKALTKDPQD YPDKKSLPHV HVALWINSQG GRKVKAGDTI SYVICQDGSN LSASQRAYAQ EQLQKQENLS IDTQYYLSQQ VHPVVARIC EPIDGIDSAL IAMWLGLDPS QFRAHRHYQQ DEENDALLGG PSQLTDEEKY RDCERFKFFC PKCGTENIYD N VFDGSGLQ IEPGLKRCSK PECDASPLDY VIQVHNKLLL DIRRYIKKYY SGWLVCEEKT CQNRTRRLPL SFSRNGPICQ AC SKATLRS EYPEKALYTQ LCFYRFIFDW DYALEKVVSE QERGHLKKKL FQESENQYKK LKSTVDQVLS RSGYSEVNLS KLF QTLNTI K

UniProtKB: DNA polymerase alpha catalytic subunit

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Macromolecule #2: DNA primase large subunit

MacromoleculeName: DNA primase large subunit / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Xenopus laevis (African clawed frog)
Molecular weightTheoretical: 59.673844 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MLFSRDRKYR HNTRLTGDRK GDLYPSSLQF YQHPPTENIS LIEFETFAIE RLKLLKAVEN LGVSYVKNSE EYSKKLELEL RKLKFPYRP LHEEISDDVY DLRRKDHISH FILRLAYCQS EDLRRWFIQQ EMDLFKFRFG LLTKESVQEF LKLNDLQYVA I SEDEKNMH ...String:
MLFSRDRKYR HNTRLTGDRK GDLYPSSLQF YQHPPTENIS LIEFETFAIE RLKLLKAVEN LGVSYVKNSE EYSKKLELEL RKLKFPYRP LHEEISDDVY DLRRKDHISH FILRLAYCQS EDLRRWFIQQ EMDLFKFRFG LLTKESVQEF LKLNDLQYVA I SEDEKNMH KEDLMNSSFG LSLTKMEDTE FYKVPFQAAL DLVRPRKVFL WRGFAFIPHK DIVSIVLNDF RAKLSKALAL SA RSLPVVQ SDERLQPLLN HLSHSYIGQD FSSQSNTGKI SLEQIDGFAA KSFPLCMRQL HKSLRENHHL RHGGRMQYGL FLK GIGLTL EQALQFWRLE FTKGKVDSEK FDKVYAYSIR HNYGKEGKRT DYTPYSCMKV ILSNPPSQGD YHGCPFRHSD PELL KQKLQ SFKVPSSGIN QILELVKGMH YQLACQKYFE LTHSVDDCGF SLNHPNQYFA ESQKLLTGSR EIKKEQTARD SPAVT ASQL SSSSSSASIP KSQSSAPEME DLEQIFSEY

UniProtKB: DNA primase large subunit

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Macromolecule #3: DNA template

MacromoleculeName: DNA template / type: dna / ID: 3 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 15.458913 KDa
SequenceString:
(DT)(DG)(DT)(DA)(DT)(DG)(DT)(DA)(DT)(DG) (DT)(DA)(DT)(DG)(DT)(DC)(DG)(DC)(DT)(DA) (DA)(DG)(DT)(DT)(DC)(DA)(DC)(DG)(DC) (DA)(DG)(DT)(DA)(DT)(DC)(DC)(DT)(DG)(DT) (DA) (DT)(DG)(DT)(DA)(DT)(DG)(DT)(DA) (DT)(DG)

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Macromolecule #4: RNA-DNA primer

MacromoleculeName: RNA-DNA primer / type: other / ID: 4 / Number of copies: 1
Classification: polydeoxyribonucleotide/polyribonucleotide hybrid
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 6.433907 KDa
SequenceString:
(GTP)GAUACUGC(DG) (DT)(DG)(DA)(DA)(DC)(DT)(DT)(DA)(DG)(DOC)

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Macromolecule #5: 2'-DEOXYGUANOSINE-5'-TRIPHOSPHATE

MacromoleculeName: 2'-DEOXYGUANOSINE-5'-TRIPHOSPHATE / type: ligand / ID: 5 / Number of copies: 1 / Formula: DGT
Molecular weightTheoretical: 507.181 Da
Chemical component information

ChemComp-DGT:
2'-DEOXYGUANOSINE-5'-TRIPHOSPHATE

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Macromolecule #6: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 6 / Number of copies: 2 / Formula: MG
Molecular weightTheoretical: 24.305 Da

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Macromolecule #7: IRON/SULFUR CLUSTER

MacromoleculeName: IRON/SULFUR CLUSTER / type: ligand / ID: 7 / Number of copies: 1 / Formula: SF4
Molecular weightTheoretical: 351.64 Da
Chemical component information

ChemComp-FS1:
IRON/SULFUR CLUSTER

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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

Concentration0.9 mg/mL
BufferpH: 7.5
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TALOS ARCTICA
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Number grids imaged: 3 / Number real images: 13641 / Average electron dose: 50.0 e/Å2
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 2.5 µm / Nominal defocus min: 1.5 µm / Nominal magnification: 45000
Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 8928051
Startup modelType of model: INSILICO MODEL / In silico model: Ab initio reconstruction
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 4.4 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION / Number images used: 42953
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial model
PDB IDChain

5f0q

source_name: PDB, initial_model_type: experimental model

8g9l

source_name: PDB, initial_model_type: experimental model

8g9n

source_name: PDB, initial_model_type: experimental model
RefinementSpace: REAL / Protocol: FLEXIBLE FIT
Output model

PDB-8g9o:
Complete DNA elongation subcomplex of Xenopus laevis DNA polymerase alpha-primase

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