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- EMDB-42035: RNA priming complex of Human Polymerase alpha-primase (Conformation 3) -

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Basic information

Entry
Database: EMDB / ID: EMD-42035
TitleRNA priming complex of Human Polymerase alpha-primase (Conformation 3)
Map data
Sample
  • Complex: Ternary complex of polymerase alpha-primase bound to an RNA primed-template substrate.
    • Protein or peptide: DNA polymerase alpha subunit BDNA polymerase
    • Protein or peptide: DNA polymerase alpha catalytic subunit
    • Protein or peptide: DNA primase large subunitPrimase
    • Protein or peptide: DNA primase small subunitPrimase
    • RNA: RNA priming substrate - primer
    • DNA: RNA priming substrate - template
KeywordsPolymerase / Primase / Polymerase Alpha / RNA priming / REPLICATION
Function / homology
Function and homology information


DNA primase AEP / positive regulation of DNA primase activity / ribonucleotide binding / DNA replication initiation / DNA/RNA hybrid binding / Telomere C-strand synthesis initiation / Inhibition of replication initiation of damaged DNA by RB1/E2F1 / Polymerase switching / Processive synthesis on the lagging strand / alpha DNA polymerase:primase complex ...DNA primase AEP / positive regulation of DNA primase activity / ribonucleotide binding / DNA replication initiation / DNA/RNA hybrid binding / Telomere C-strand synthesis initiation / Inhibition of replication initiation of damaged DNA by RB1/E2F1 / Polymerase switching / Processive synthesis on the lagging strand / alpha DNA polymerase:primase complex / regulation of type I interferon production / Removal of the Flap Intermediate / DNA primase activity / Polymerase switching on the C-strand of the telomere / lagging strand elongation / DNA replication, synthesis of primer / mitotic DNA replication initiation / DNA strand elongation involved in DNA replication / leading strand elongation / DNA synthesis involved in DNA repair / G1/S-Specific Transcription / DNA replication origin binding / DNA replication initiation / Activation of the pre-replicative complex / Defective pyroptosis / nuclear matrix / double-strand break repair via nonhomologous end joining / protein import into nucleus / single-stranded DNA binding / nuclear envelope / 4 iron, 4 sulfur cluster binding / DNA replication / DNA-directed DNA polymerase / DNA-directed DNA polymerase activity / nucleotide binding / DNA repair / chromatin binding / chromatin / nucleolus / protein kinase binding / magnesium ion binding / DNA binding / zinc ion binding / nucleoplasm / membrane / metal ion binding / nucleus / cytosol
Similarity search - Function
DNA polymerase alpha, subunit B, N-terminal domain superfamily / 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, subunit B, N-terminal domain superfamily / 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 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-directed DNA polymerase, family B, multifunctional 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 / negative staining / Resolution: 7.41 Å
AuthorsCordoba JJ / Chazin WJ
Funding support United States, 3 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)2T32GM008320-31 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R35GM118089 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R35GM136401 United States
CitationJournal: J Mol Biol / Year: 2023
Title: Flexibility and Distributive Synthesis Regulate RNA Priming and Handoff in Human DNA Polymerase α-Primase.
Authors: John J Cordoba / Elwood A Mullins / Lauren E Salay / Brandt F Eichman / Walter J Chazin /
Abstract: DNA replication in eukaryotes relies on the synthesis of a ∼30-nucleotide RNA/DNA primer strand through the dual action of the heterotetrameric polymerase α-primase (pol-prim) enzyme. Synthesis of ...DNA replication in eukaryotes relies on the synthesis of a ∼30-nucleotide RNA/DNA primer strand through the dual action of the heterotetrameric polymerase α-primase (pol-prim) enzyme. Synthesis of the 7-10-nucleotide RNA primer is regulated by the C-terminal domain of the primase regulatory subunit (PRIM2C) and is followed by intramolecular handoff of the primer to pol α for extension by ∼20 nucleotides of DNA. Here, we provide evidence that RNA primer synthesis is governed by a combination of the high affinity and flexible linkage of the PRIM2C domain and the surprisingly low affinity of the primase catalytic domain (PRIM1) for substrate. Using a combination of small angle X-ray scattering and electron microscopy, we found significant variability in the organization of PRIM2C and PRIM1 in the absence and presence of substrate, and that the population of structures with both PRIM2C and PRIM1 in a configuration aligned for synthesis is low. Crosslinking was used to visualize the orientation of PRIM2C and PRIM1 when engaged by substrate as observed by electron microscopy. Microscale thermophoresis was used to measure substrate affinities for a series of pol-prim constructs, which showed that the PRIM1 catalytic domain does not bind the template or emergent RNA-primed templates with appreciable affinity. Together, these findings support a model of RNA primer synthesis in which generation of the nascent RNA strand and handoff of the RNA-primed template from primase to polymerase α is mediated by the high degree of inter-domain flexibility of pol-prim, the ready dissociation of PRIM1 from its substrate, and the much higher affinity of the POLA1cat domain of polymerase α for full-length RNA-primed templates.
History
DepositionSep 19, 2023-
Header (metadata) releaseNov 15, 2023-
Map releaseNov 15, 2023-
UpdateNov 22, 2023-
Current statusNov 22, 2023Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_42035.png

Downloads & links

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Map

FileDownload / File: emd_42035.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 1.79015 Å
Density
Contour LevelBy AUTHOR: 1.81
Minimum - Maximum-1.0159925 - 4.118256
Average (Standard dev.)-0.01985491 (±0.16365242)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 458.2784 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #1

Fileemd_42035_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_42035_half_map_2.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Ternary complex of polymerase alpha-primase bound to an RNA prime...

EntireName: Ternary complex of polymerase alpha-primase bound to an RNA primed-template substrate.
Components
  • Complex: Ternary complex of polymerase alpha-primase bound to an RNA primed-template substrate.
    • Protein or peptide: DNA polymerase alpha subunit BDNA polymerase
    • Protein or peptide: DNA polymerase alpha catalytic subunit
    • Protein or peptide: DNA primase large subunitPrimase
    • Protein or peptide: DNA primase small subunitPrimase
    • RNA: RNA priming substrate - primer
    • DNA: RNA priming substrate - template

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Supramolecule #1: Ternary complex of polymerase alpha-primase bound to an RNA prime...

SupramoleculeName: Ternary complex of polymerase alpha-primase bound to an RNA primed-template substrate.
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all / Details: Complex stabilized by BS3 crosslinking.
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 200 KDa

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Macromolecule #1: DNA polymerase alpha subunit B

MacromoleculeName: DNA polymerase alpha subunit B / type: protein_or_peptide / ID: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: GSATPSQKYN SRSNRGEVVT SFGLAQGV S WSGRGGAGNI SLKVLGCPEA LTGSYKSMFQ KLPDIREVLT CKIEELGSEL KEHYKIEAF TPLLAPAQEP VTLLGQIGCD SNGKLNNKSV ILEGDREHSS GAQIPVDLSE LKEYSLFPGQ VVIMEGINT TGRKLVATKL ...String:
GSATPSQKYN SRSNRGEVVT SFGLAQGV S WSGRGGAGNI SLKVLGCPEA LTGSYKSMFQ KLPDIREVLT CKIEELGSEL KEHYKIEAF TPLLAPAQEP VTLLGQIGCD SNGKLNNKSV ILEGDREHSS GAQIPVDLSE LKEYSLFPGQ VVIMEGINT TGRKLVATKL YEGVPLPFYQ PTEEDADFEQ SMVLVACGPY TTSDSITYDP L LDLIAVIN HDRPDVCILF GPFLDAKHEQ VENCLLTSPF EDIFKQCLRT IIEGTRSSGS HL VFVPSLR DVHHEPVYPQ PPFSYSDLSR EDKKQVQFVS EPCSLSINGV IFGLTSTDLL FHL GAEEIS SSSGTSDRFS RILKHILTQR SYYPLYPPQE DMAIDYESFY VYAQLPVTPD VLII PSELR YFVKDVLGCV CVNPGRLTKG QVGGTFARLY LRRPAADGAE RQSPCIAVQV VRI

UniProtKB: DNA polymerase alpha subunit B

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Macromolecule #2: DNA polymerase alpha catalytic subunit

MacromoleculeName: DNA polymerase alpha catalytic subunit / type: protein_or_peptide / ID: 2 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Escherichia coli BL21 (bacteria)
SequenceString: MAQLTDEEKY RDCERFKCPC PTCGTENIYD NVFDGSGTDM EPSLYRCSNI DCKASPL TF TVQLSNKLIM DIRRFIKKYY DGWLICEEPT CRNRTRHLPL QFSRTGPLCP ACMKATLQ P EYSDKSLYTQ LCFYRYIFDA ECALEKLTTD HEKDKLKKQF FTPKVLQDYR ...String:
MAQLTDEEKY RDCERFKCPC PTCGTENIYD NVFDGSGTDM EPSLYRCSNI DCKASPL TF TVQLSNKLIM DIRRFIKKYY DGWLICEEPT CRNRTRHLPL QFSRTGPLCP ACMKATLQ P EYSDKSLYTQ LCFYRYIFDA ECALEKLTTD HEKDKLKKQF FTPKVLQDYR KLKNTAEQF LSRSGYSEVN LSKLFAGCAV KS

UniProtKB: DNA polymerase alpha catalytic subunit

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Macromolecule #3: DNA primase large subunit

MacromoleculeName: DNA primase large subunit / type: protein_or_peptide / ID: 3 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MEFSGRKWRK LRLAGDQRNA SYPHCLQFYL QPPSENISLI EFENLAIDRV KLLKSVENLG VSYVKGTEQ YQSKLESELR KLKFSYRENL EDEYEPRRRD HISHFILRLA YCQSEELRRW F IQQEMDLL RFRFSILPKD KIQDFLKDSQ LQFEAISDEE KTLREQEIVA ...String:
MEFSGRKWRK LRLAGDQRNA SYPHCLQFYL QPPSENISLI EFENLAIDRV KLLKSVENLG VSYVKGTEQ YQSKLESELR KLKFSYRENL EDEYEPRRRD HISHFILRLA YCQSEELRRW F IQQEMDLL RFRFSILPKD KIQDFLKDSQ LQFEAISDEE KTLREQEIVA SSPSLSGLKL GF ESIYKIP FADALDLFRG RKVYLEDGFA YVPLKDIVAI ILNEFRAKLS KALALTARSL PAV QSDERL QPLLNHLSHS YTGQDYSTQG NVGKISLDQI DLLSTKSFPP CMRQLHKALR ENHH LRHGG RMQYGLFLKG IGLTLEQALQ FWKQEFIKGK MDPDKFDKGY SYNIRHSFGK EGKRT DYTP FSCLKIILSN PPSQGDYHGC PFRHSDPELL KQKLQSYKIS PGGISQILDL VKGTHY QVA CQKYFEMIHN VDDCGFSLNH PNQFFCESQR ILNGGKDIKK EPIQPETPQP KPSVQKT KD ASSALASLNS SLEMDMEGLE DYFSEDS

UniProtKB: DNA primase large subunit

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Macromolecule #4: DNA primase small subunit

MacromoleculeName: DNA primase small subunit / type: protein_or_peptide / ID: 4 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Escherichia coli BL21 (bacteria)
SequenceString: GSMETFDPTE LPELLKLYYR RLFPYSQYYR WLNYGGVIKN YFQHREFSFT LKDDIYIRYQ SF NNQSDLE KEMQKMNPYK IDIGAVYSHR PNQHNTVKLG AFQAQEKELV FDIDMTDYDD VRR CCSSAD ICPKCWTLMT MAIRIIDRAL KEDFGFKHRL WVYSGRRGVH ...String:
GSMETFDPTE LPELLKLYYR RLFPYSQYYR WLNYGGVIKN YFQHREFSFT LKDDIYIRYQ SF NNQSDLE KEMQKMNPYK IDIGAVYSHR PNQHNTVKLG AFQAQEKELV FDIDMTDYDD VRR CCSSAD ICPKCWTLMT MAIRIIDRAL KEDFGFKHRL WVYSGRRGVH CWVCDESVRK LSSA VRSGI VEYLSLVKGG QDVKKKVHLS EKIHPFIRKS INIIKKYFEE YALVNQDILE NKESW DKIL ALVPETIHDE LQQSFQKSHN SLQRWEHLKK VASRYQNNIK NDKYGPWLEW EIMLQY CFP RLDINVSKGI NHLLKSPFSV HPKTGRISVP IDLQKVDQFD PFTVPTISFI CRELDAI ST NEEEKEENEA ESDVKHRTRD YKKTSLAPYV KVFEHFLENL DKSRKGELLK KSDLQKDF

UniProtKB: DNA primase small subunit

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Macromolecule #5: RNA priming substrate - primer

MacromoleculeName: RNA priming substrate - primer / type: rna / ID: 5 / Details: 5' triphosphorylated
Source (natural)Organism: Homo sapiens (human)
SequenceString:
GGAUACUG

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Macromolecule #6: RNA priming substrate - template

MacromoleculeName: RNA priming substrate - template / type: dna / ID: 6 / Classification: DNA
Source (natural)Organism: Homo sapiens (human)
SequenceString:
GTATGTATGT CAGTATCCTG TATGTATGA

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Experimental details

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Structure determination

Methodnegative staining
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration0.009 mg/mL
BufferpH: 7.5
Component:
ConcentrationFormulaName
150.0 mMNaClSodium chlorideSodium chloride
20.0 mMC8H18N2O4SHEPES
5.0 mMMgCl2Magnesium chloride
1.0 mMC9H15O6PTCEP
StainingType: NEGATIVE / Material: Uranyl Formate
Details: 2.5 uL sample was applied to grid for 1 minute, then blotted with filter paper, washed for 5 seconds with DI water, blotted, washed for 5 seconds in stain, blotted, then stained for 90 seconds.

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Electron microscopy

MicroscopeFEI TECNAI 20
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: SPOT SCAN / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 1.2 mm / Nominal defocus max: 1.5 µm / Nominal defocus min: 1.5 µm
Image recordingFilm or detector model: GATAN ULTRASCAN 4000 (4k x 4k) / Digitization - Dimensions - Width: 4096 pixel / Digitization - Dimensions - Height: 4096 pixel / Number grids imaged: 1 / Number real images: 1083 / Average electron dose: 20.0 e/Å2

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Image processing

Particle selectionNumber selected: 1628779
Startup modelType of model: NONE
Initial angle assignmentType: NOT APPLICABLE
Final 3D classificationNumber classes: 3 / Avg.num./class: 16500 / Software - Name: cryoSPARC (ver. 4.1)
Final angle assignmentType: NOT APPLICABLE
Final reconstructionNumber classes used: 1 / Resolution.type: BY AUTHOR / Resolution: 7.41 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 1.4) / Number images used: 14867
FSC plot (resolution estimation)

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