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Yorodumi- EMDB-17033: Pol I bound to extended and displaced DNA section - open conformation -
+Open data
-Basic information
Entry | Database: EMDB / ID: EMD-17033 | |||||||||
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Title | Pol I bound to extended and displaced DNA section - open conformation | |||||||||
Map data | ||||||||||
Sample |
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Keywords | DNA Polymerase I / Okazaki fragment maturation / DNA BINDING PROTEIN | |||||||||
Function / homology | Function and homology information 5'-3' exonuclease activity / 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 ...5'-3' exonuclease activity / 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 / cytosol / cytoplasm Similarity search - Function | |||||||||
Biological species | Escherichia coli (E. coli) / Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria) / synthetic construct (others) | |||||||||
Method | single particle reconstruction / cryo EM / Resolution: 4.0 Å | |||||||||
Authors | Botto M / Borsellini A / Lamers MH | |||||||||
Funding support | 1 items
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Citation | Journal: 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 |
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-Structure visualization
Supplemental images |
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-Downloads & links
-EMDB archive
Map data | emd_17033.map.gz | 4.9 MB | EMDB map data format | |
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Header (meta data) | emd-17033-v30.xml emd-17033.xml | 16.3 KB 16.3 KB | Display Display | EMDB header |
Images | emd_17033.png | 62.8 KB | ||
Filedesc metadata | emd-17033.cif.gz | 5.9 KB | ||
Others | emd_17033_half_map_1.map.gz emd_17033_half_map_2.map.gz | 49.6 MB 49.5 MB | ||
Archive directory | http://ftp.pdbj.org/pub/emdb/structures/EMD-17033 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-17033 | HTTPS FTP |
-Validation report
Summary document | emd_17033_validation.pdf.gz | 606.6 KB | Display | EMDB validaton report |
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Full document | emd_17033_full_validation.pdf.gz | 606.2 KB | Display | |
Data in XML | emd_17033_validation.xml.gz | 12.1 KB | Display | |
Data in CIF | emd_17033_validation.cif.gz | 14.2 KB | Display | |
Arichive directory | https://ftp.pdbj.org/pub/emdb/validation_reports/EMD-17033 ftp://ftp.pdbj.org/pub/emdb/validation_reports/EMD-17033 | HTTPS FTP |
-Related structure data
Related structure data | 8ooyMC 8oo6C M: atomic model generated by this map C: citing same article (ref.) |
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Similar structure data | Similarity search - Function & homologyF&H Search |
-Links
EMDB pages | EMDB (EBI/PDBe) / EMDataResource |
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Related items in Molecule of the Month |
-Map
File | Download / File: emd_17033.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) | ||||||||||||||||||||||||||||||||||||
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Projections & slices | Image control
Images are generated by Spider. | ||||||||||||||||||||||||||||||||||||
Voxel size | X=Y=Z: 0.836 Å | ||||||||||||||||||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||||||||||||||||||
Details | EMDB XML:
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-Supplemental data
-Half map: #2
File | emd_17033_half_map_1.map | ||||||||||||
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Projections & Slices |
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Density Histograms |
-Half map: #1
File | emd_17033_half_map_2.map | ||||||||||||
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Projections & Slices |
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Density Histograms |
-Sample components
-Entire : Escherichia coli DNA Polymerase I in complex with DNA
Entire | Name: Escherichia coli DNA Polymerase I in complex with DNA |
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Components |
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-Supramolecule #1: Escherichia coli DNA Polymerase I in complex with DNA
Supramolecule | Name: Escherichia coli DNA Polymerase I in complex with DNA / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#4 |
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Source (natural) | Organism: Escherichia coli (E. coli) |
-Macromolecule #1: DNA polymerase I
Macromolecule | Name: DNA polymerase I / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO / EC number: DNA-directed DNA polymerase |
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Source (natural) | Organism: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria) |
Molecular weight | Theoretical: 67.991461 KDa |
Recombinant expression | Organism: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria) |
Sequence | String: 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 |
-Macromolecule #2: Template DNA
Macromolecule | Name: Template DNA / type: other / ID: 2 / Number of copies: 1 Classification: polydeoxyribonucleotide/polyribonucleotide hybrid |
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Source (natural) | Organism: synthetic construct (others) |
Molecular weight | Theoretical: 8.374174 KDa |
Sequence | String: AACG(DT)CG(DT)GA C(DT)GGGAAAAC CC(DT)GGC |
-Macromolecule #3: Extending Primer
Macromolecule | Name: Extending Primer / type: other / ID: 3 / Number of copies: 1 Classification: polydeoxyribonucleotide/polyribonucleotide hybrid |
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Source (natural) | Organism: synthetic construct (others) |
Molecular weight | Theoretical: 5.691538 KDa |
Sequence | String: GCCAGGG(DT)(DT)(DT) (DT)CCCAG(DT)C |
-Macromolecule #4: Displacing Primer
Macromolecule | Name: Displacing Primer / type: dna / ID: 4 / Number of copies: 1 / Classification: DNA |
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Source (natural) | Organism: synthetic construct (others) |
Molecular weight | Theoretical: 2.11341 KDa |
Sequence | String: (DC)(DG)(DA)(DC)(DG)(DT)(DT) |
-Macromolecule #5: MAGNESIUM ION
Macromolecule | Name: MAGNESIUM ION / type: ligand / ID: 5 / Number of copies: 1 / Formula: MG |
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Molecular weight | Theoretical: 24.305 Da |
-Experimental details
-Structure determination
Method | cryo EM |
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Processing | single particle reconstruction |
Aggregation state | particle |
-Sample preparation
Buffer | pH: 8.5 |
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Sugar embedding | Material: water |
Vitrification | Cryogen name: ETHANE / Instrument: LEICA PLUNGER |
-Electron microscopy
Microscope | FEI TITAN KRIOS |
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Image recording | Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Average electron dose: 40.0 e/Å2 |
Electron beam | Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN |
Electron optics | Illumination 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 |
-Image processing
Startup model | Type of model: PDB ENTRY PDB model - PDB ID: |
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Final reconstruction | Resolution.type: BY AUTHOR / Resolution: 4.0 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 164102 |
Initial angle assignment | Type: PROJECTION MATCHING |
Final angle assignment | Type: ANGULAR RECONSTITUTION |