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- EMDB-29886: Partial DNA termination subcomplex of Xenopus laevis DNA polymera... -

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Basic information

Entry
Database: EMDB / ID: EMD-29886
TitlePartial DNA termination subcomplex of Xenopus laevis DNA polymerase alpha-primase
Map dataPartial DNA termination subcomplex of Xenopus laevis DNA polymerase alpha-primase
Sample
  • Complex: Polymerase alpha-primase with a DNA termination substrate
    • Complex: Polymerase alpha
      • Protein or peptide: POLA1
    • Complex: Primase
    • Complex: DNA termination substrate
      • DNA: DNA template
      • DNA: RNA-DNA primer
Biological speciesXenopus laevis (African clawed frog) / synthetic construct (others)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.9 Å
AuthorsMullins EA / 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)CA92584 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 22, 2023-
Header (metadata) releaseApr 12, 2023-
Map releaseApr 12, 2023-
UpdateApr 12, 2023-
Current statusApr 12, 2023Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_29886.png

Downloads & links

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Map

FileDownload / File: emd_29886.map.gz / Format: CCP4 / Size: 244.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationPartial DNA termination subcomplex of Xenopus laevis DNA polymerase alpha-primase
Voxel sizeX=Y=Z: 0.82 Å
Density
Contour LevelBy AUTHOR: 0.2
Minimum - Maximum-0.28128654 - 0.8381235
Average (Standard dev.)-0.0014583338 (±0.024696702)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions400400400
Spacing400400400
CellA=B=C: 328.0 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: Partial DNA termination subcomplex of Xenopus laevis DNA...

Fileemd_29886_half_map_1.map
AnnotationPartial DNA termination 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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Half map: Partial DNA termination subcomplex of Xenopus laevis DNA...

Fileemd_29886_half_map_2.map
AnnotationPartial DNA termination 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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Sample components

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Entire : Polymerase alpha-primase with a DNA termination substrate

EntireName: Polymerase alpha-primase with a DNA termination substrate
Components
  • Complex: Polymerase alpha-primase with a DNA termination substrate
    • Complex: Polymerase alpha
      • Protein or peptide: POLA1
    • Complex: Primase
    • Complex: DNA termination substrate
      • DNA: DNA template
      • DNA: RNA-DNA primer

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

SupramoleculeName: Polymerase alpha-primase with a DNA termination substrate
type: complex / ID: 1 / Chimera: Yes / Parent: 0 / Macromolecule list: all
Details: Heterotetrameric protein complex with an RNA-DNA/DNA duplex
Molecular weightTheoretical: 330 KDa

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Supramolecule #2: Polymerase alpha

SupramoleculeName: Polymerase alpha / type: complex / ID: 2 / Chimera: Yes / Parent: 1 / Macromolecule list: #1 / Details: Heterodimer consisting of POLA1 and POLA2
Source (natural)Organism: Xenopus laevis (African clawed frog)
Molecular weightTheoretical: 200 KDa

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Supramolecule #3: Primase

SupramoleculeName: Primase / type: complex / ID: 3 / Chimera: Yes / Parent: 1 / Details: Heterodimer consisting of PRIM1 and PRIM2
Source (natural)Organism: Xenopus laevis (African clawed frog)
Molecular weightTheoretical: 110 KDa

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Supramolecule #4: DNA termination substrate

SupramoleculeName: DNA termination substrate / type: complex / ID: 4 / Chimera: Yes / Parent: 1 / Macromolecule list: #2-#3 / Details: RNA-DNA/DNA duplex
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 30 KDa

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Macromolecule #1: POLA1

MacromoleculeName: POLA1 / type: protein_or_peptide / ID: 1 / Enantiomer: LEVO
Source (natural)Organism: Xenopus laevis (African clawed frog)
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: SNAADGSQVF RFYWLDAYED QYSQPGVVYL FGKVWIESAD AYVSCCVSVK NIERTVYLLP RENRVQLSTG KDTGAPVSMM HVYQEFNEAV AEKYKIMKFK SKKVDKDYAF EIPDVPASSE YLEVRYSADS PQLPQDLKGE TFSHVFGTNT SSLELFLLSR KIKGPSWLEI ...String:
SNAADGSQVF RFYWLDAYED QYSQPGVVYL FGKVWIESAD AYVSCCVSVK NIERTVYLLP RENRVQLSTG KDTGAPVSMM HVYQEFNEAV AEKYKIMKFK SKKVDKDYAF EIPDVPASSE YLEVRYSADS PQLPQDLKGE TFSHVFGTNT SSLELFLLSR KIKGPSWLEI KSPQLSSQPM SWCKVEAVVT RPDQVSVVKD LAPPPVVVLS LSMKTVQNAK THQNEIVAIA ALVHHTFPLD KAPPQPPFQT HFCVLSKLND CIFPYDYNEA VKQKNANIEI ALTERTLLGF FLAKIHKIDP DVIVGHDIYG FDLEVLLQRI NSCKVPFWSK IGRLRRSVMP KLGGRSGFAE RNAACGRIIC DIEISAKELI RCKSYHLSEL VHQILKAERV VIPPENIRNA YNDSVHLLYM LENTWIDAKF ILQIMCELNV LPLALQITNI AGNVMSRTLM GGRSERNEYL LLHAFTENNF IVPDKPVFKK MQQTTVEDND DMGTDQNKNK SRKKAAYAGG LVLEPKVGFY DKFILLLDFN SLYPSIIQEY NICFTTVHRE APSTQKGEDQ DEIPELPHSD LEMGILPREI RKLVERRRHV KQLMKQPDLN PDLYLQYDIR QKALKLTANS MYGCLGFSYS RFYAKPLAAL VTHQGREILL HTKEMVQKMN LEVIYGDTDS IMINTNCNNL EEVFKLGNRV KSEINKSYKL LEIDIDGIFK SLLLLKKKKY AALTVEPTGD GKYVTKQELK GLDIVRRDWC ELAKQAGNYV ISQILSDQPR DSIVENIQKK LTEIGENVTN GTVPITQYEI NKALTKDPQD YPDKKSLPHV HVALWINSQG GRKVKAGDTI SYVICQDGSN LSASQRAYAQ EQLQKQENLS IDTQYYLSQQ VHPVVARICE PIDGIDSALI AMWLGLDPSQ FRAHRHYQQD EENDALLGGP SQLTDEEKYR DCERFKFFCP KCGTENIYDN VFDGSGLQIE PGLKRCSKPE CDASPLDYVI QVHNKLLLDI RRYIKKYYSG WLVCEEKTCQ NRTRRLPLSF SRNGPICQAC SKATLRSEYP EKALYTQLCF YRFIFDWDYA LEKVVSEQER GHLKKKLFQE SENQYKKLKS TVDQVLSRSG YSEVNLSKLF QTLNTIK

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Macromolecule #2: DNA template

MacromoleculeName: DNA template / type: dna / ID: 2 / Classification: DNA
Source (natural)Organism: synthetic construct (others)
SequenceString: (DT)(DG)(DT)(DA)(DT)(DG)(DT)(DA)(DT)(DG) (DT)(DA)(DT)(DG)(DT)(DC)(DG)(DC)(DT)(DA) (DC)(DA)(DA)(DT)(DC)(DG)(DC)(DT)(DA)(DA) (DG)(DT)(DT)(DC)(DA)(DC)(DG)(DC)(DA)(DG) (DT)(DA)(DT)(DC)(DC)(DT)(DG) ...String:
(DT)(DG)(DT)(DA)(DT)(DG)(DT)(DA)(DT)(DG) (DT)(DA)(DT)(DG)(DT)(DC)(DG)(DC)(DT)(DA) (DC)(DA)(DA)(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 #3: RNA-DNA primer

MacromoleculeName: RNA-DNA primer / type: dna / ID: 3 / Classification: DNA
Source (natural)Organism: synthetic construct (others)
SequenceString:
(GTP)GAUACUGC(DG) (DT)(DG)(DA)(DA)(DC)(DT)(DT)(DA)(DG)(DC) (DG)(DA)(DT)(DT)(DG)(DT)(DA)(DG)(DOC)

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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 TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 2.0 µm / Nominal defocus min: 0.8 µm / Nominal magnification: 105000
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Number grids imaged: 1 / Number real images: 22893 / Average electron dose: 55.8 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 25216892
Startup modelType of model: INSILICO MODEL / In silico model: Ab initio reconstruction
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 3.9 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC / Number images used: 278995
FSC plot (resolution estimation)

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