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- EMDB-17033: Pol I bound to extended and displaced DNA section - open conformation -

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Basic information

Entry
Database: EMDB / ID: EMD-17033
TitlePol I bound to extended and displaced DNA section - open conformation
Map data
Sample
  • Complex: Escherichia coli DNA Polymerase I in complex with DNA
    • Protein or peptide: DNA polymerase I
    • Other: Template DNA
    • Other: Extending Primer
    • DNA: Displacing Primer
  • Ligand: MAGNESIUM ION
KeywordsDNA Polymerase I / Okazaki fragment maturation / DNA BINDING PROTEIN
Function / homology
Function and homology information


5'-3' exonuclease activity / double-strand break repair via alternative nonhomologous end joining / 3'-5' exonuclease activity / base-excision repair / DNA-templated DNA replication / double-strand break repair / DNA replication / DNA-directed DNA polymerase / DNA-directed DNA polymerase activity / DNA repair ...5'-3' exonuclease activity / double-strand break repair via alternative nonhomologous end joining / 3'-5' exonuclease activity / base-excision repair / DNA-templated DNA replication / double-strand break repair / DNA replication / DNA-directed DNA polymerase / DNA-directed DNA polymerase activity / DNA repair / DNA binding / cytoplasm / cytosol
Similarity search - Function
3'-5' exonuclease / DNA polymerase I-like, H3TH domain / 5'-3' exonuclease, C-terminal SAM fold / 5'-3' exonuclease, alpha-helical arch, N-terminal / 5'-3' exonuclease, N-terminal resolvase-like domain / 5'-3' exonuclease / 5'-3' exonuclease / DNA polymerase 1 / 3'-5' exonuclease / 3'-5' exonuclease domain ...3'-5' exonuclease / DNA polymerase I-like, H3TH domain / 5'-3' exonuclease, C-terminal SAM fold / 5'-3' exonuclease, alpha-helical arch, N-terminal / 5'-3' exonuclease, N-terminal resolvase-like domain / 5'-3' exonuclease / 5'-3' exonuclease / DNA polymerase 1 / 3'-5' exonuclease / 3'-5' exonuclease domain / Helix-hairpin-helix motif, class 2 / Helix-hairpin-helix class 2 (Pol1 family) motifs / 5'-3' exonuclease, C-terminal domain superfamily / DNA polymerase A / DNA polymerase family A / DNA-directed DNA polymerase, family A, conserved site / DNA polymerase family A signature. / DNA-directed DNA polymerase, family A, palm domain / DNA polymerase A domain / PIN-like domain superfamily / Ribonuclease H superfamily / Ribonuclease H-like superfamily / DNA/RNA polymerase superfamily
Similarity search - Domain/homology
Biological speciesEscherichia coli (E. coli) / Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria) / synthetic construct (others)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.0 Å
AuthorsBotto M / Borsellini A / Lamers MH
Funding support1 items
OrganizationGrant numberCountry
Other government
CitationJournal: Nat Struct Mol Biol / Year: 2023
Title: A four-point molecular handover during Okazaki maturation.
Authors: Margherita M Botto / Alessandro Borsellini / Meindert H Lamers /
Abstract: DNA replication introduces thousands of RNA primers into the lagging strand that need to be removed for replication to be completed. In Escherichia coli when the replicative DNA polymerase Pol IIIα ...DNA replication introduces thousands of RNA primers into the lagging strand that need to be removed for replication to be completed. In Escherichia coli when the replicative DNA polymerase Pol IIIα terminates at a previously synthesized RNA primer, DNA Pol I takes over and continues DNA synthesis while displacing the downstream RNA primer. The displaced primer is subsequently excised by an endonuclease, followed by the sealing of the nick by a DNA ligase. Yet how the sequential actions of Pol IIIα, Pol I polymerase, Pol I endonuclease and DNA ligase are coordinated is poorly defined. Here we show that each enzymatic activity prepares the DNA substrate for the next activity, creating an efficient four-point molecular handover. The cryogenic-electron microscopy structure of Pol I bound to a DNA substrate with both an upstream and downstream primer reveals how it displaces the primer in a manner analogous to the monomeric helicases. Moreover, we find that in addition to its flap-directed nuclease activity, the endonuclease domain of Pol I also specifically cuts at the RNA-DNA junction, thus marking the end of the RNA primer and creating a 5' end that is a suitable substrate for the ligase activity of LigA once all RNA has been removed.
History
DepositionApr 6, 2023-
Header (metadata) releaseAug 9, 2023-
Map releaseAug 9, 2023-
UpdateNov 1, 2023-
Current statusNov 1, 2023Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_17033.png

Downloads & links

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Map

FileDownload / File: emd_17033.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 0.836 Å
Density
Contour LevelBy AUTHOR: 0.0022
Minimum - Maximum-0.027238669 - 0.05383265
Average (Standard dev.)0.00010059201 (±0.0012358373)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 214.016 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #2

Fileemd_17033_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_17033_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 : Escherichia coli DNA Polymerase I in complex with DNA

EntireName: Escherichia coli DNA Polymerase I in complex with DNA
Components
  • Complex: Escherichia coli DNA Polymerase I in complex with DNA
    • Protein or peptide: DNA polymerase I
    • Other: Template DNA
    • Other: Extending Primer
    • DNA: Displacing Primer
  • Ligand: MAGNESIUM ION

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Supramolecule #1: Escherichia coli DNA Polymerase I in complex with DNA

SupramoleculeName: Escherichia coli DNA Polymerase I in complex with DNA / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#4
Source (natural)Organism: Escherichia coli (E. coli)

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Macromolecule #1: DNA polymerase I

MacromoleculeName: DNA polymerase I / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO / EC number: DNA-directed DNA polymerase
Source (natural)Organism: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
Molecular weightTheoretical: 67.991461 KDa
Recombinant expressionOrganism: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
SequenceString: GPHDNYVTIL DEETLKAWIA KLEKAPVFAF DTETDSLDNI SANLVGLSFA IEPGVAAYIP VAHDYLDAPD QISRERALEL LKPLLEDEK ALKVGQNLKY DRGILANYGI ELRGIAFDTM LESYILNSVA GRHDMDSLAE RWLKHKTITF EEIAGKGKNQ L TFNQIALE ...String:
GPHDNYVTIL DEETLKAWIA KLEKAPVFAF DTETDSLDNI SANLVGLSFA IEPGVAAYIP VAHDYLDAPD QISRERALEL LKPLLEDEK ALKVGQNLKY DRGILANYGI ELRGIAFDTM LESYILNSVA GRHDMDSLAE RWLKHKTITF EEIAGKGKNQ L TFNQIALE EAGRYAAEDA DVTLQLHLKM WPDLQKHKGP LNVFENIEMP LVPVLSRIER NGVKIDPKVL HNHSEELTLR LA ELEKKAH EIAGEEFNLS STKQLQTILF EKQGIKPLKK TPGGAPSTSE EVLEELALDY PLPKVILEYR GLAKLKSTYT DKL PLMINP KTGRVHTSYH QAVTATGRLS STDPNLQNIP VRNEEGRRIR QAFIAPEDYV IVSADYSQIE LRIMAHLSRD KGLL TAFAE GKDIHRATAA EVFGLPLETV TSEQRRSAKA INFGLIYGMS AFGLARQLNI PRKEAQKYMD LYFERYPGVL EYMER TRAQ AKEQGYVETL DGRRLYLPDI KSSNGARRAA AERAAINAPM QGTAADIIKR AMIAVDAWLQ AEQPRVRMIM QVHDEL VFE VHKDDVDAVA KQIHQLMENC TRLDVPLLVE VGSGENWDQA H

UniProtKB: DNA polymerase I

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Macromolecule #2: Template DNA

MacromoleculeName: Template DNA / type: other / ID: 2 / Number of copies: 1
Classification: polydeoxyribonucleotide/polyribonucleotide hybrid
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 8.374174 KDa
SequenceString:
AACG(DT)CG(DT)GA C(DT)GGGAAAAC CC(DT)GGC

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Macromolecule #3: Extending Primer

MacromoleculeName: Extending Primer / type: other / ID: 3 / Number of copies: 1
Classification: polydeoxyribonucleotide/polyribonucleotide hybrid
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 5.691538 KDa
SequenceString:
GCCAGGG(DT)(DT)(DT) (DT)CCCAG(DT)C

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Macromolecule #4: Displacing Primer

MacromoleculeName: Displacing Primer / type: dna / ID: 4 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 2.11341 KDa
SequenceString:
(DC)(DG)(DA)(DC)(DG)(DT)(DT)

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Macromolecule #5: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 5 / Number of copies: 1 / Formula: MG
Molecular weightTheoretical: 24.305 Da

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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: 8.5
Sugar embeddingMaterial: water
VitrificationCryogen name: ETHANE / Instrument: LEICA PLUNGER

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Average electron dose: 40.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.5 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: PDB ENTRY
PDB model - PDB ID:

1kln

Final reconstructionResolution.type: BY AUTHOR / Resolution: 4.0 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 164102
Initial angle assignmentType: PROJECTION MATCHING
Final angle assignmentType: ANGULAR RECONSTITUTION

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