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- PDB-8oo6: Pol I bound to extended and displaced DNA section - closed confor... -
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Open data
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Basic information
Entry | Database: PDB / ID: 8oo6 | ||||||
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Title | Pol I bound to extended and displaced DNA section - closed conformation | ||||||
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![]() | DNA BINDING PROTEIN / DNA Polymerase I / Okazaki fragment maturation | ||||||
Function / homology | ![]() 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 | ||||||
Biological species | Escherichia coli 'BL21-GoldpLysS AG' synthetic construct (others) | ||||||
Method | ELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4.3 Å | ||||||
![]() | Botto, M. / Borsellini, A. / Lamers, M.H. | ||||||
Funding support | 1items
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![]() | ![]() 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
Structure viewer | Molecule: ![]() ![]() |
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Downloads & links
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Download
PDBx/mmCIF format | ![]() | 147.8 KB | Display | ![]() |
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PDB format | ![]() | 107.6 KB | Display | ![]() |
PDBx/mmJSON format | ![]() | Tree view | ![]() | |
Others | ![]() |
-Validation report
Summary document | ![]() | 952.3 KB | Display | ![]() |
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Full document | ![]() | 962 KB | Display | |
Data in XML | ![]() | 30.1 KB | Display | |
Data in CIF | ![]() | 42 KB | Display | |
Arichive directory | ![]() ![]() | HTTPS FTP |
-Related structure data
Related structure data | ![]() 17005MC ![]() 8ooyC M: map data used to model this data C: citing same article ( |
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Similar structure data | Similarity search - Function & homology ![]() |
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Links
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Assembly
Deposited unit | ![]()
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Components
#1: Protein | Mass: 67991.461 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) ![]() ![]() Gene: polA, resA, b3863, JW3835 Production host: ![]() ![]() References: UniProt: P00582, DNA-directed DNA polymerase |
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#2: DNA chain | Mass: 8311.362 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others) |
#3: DNA chain | Mass: 5483.540 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others) |
#4: DNA chain | Mass: 2755.823 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others) |
#5: Chemical | ChemComp-MG / |
Has ligand of interest | N |
-Experimental details
-Experiment
Experiment | Method: ELECTRON MICROSCOPY |
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EM experiment | Aggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction |
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Sample preparation
Component | Name: Pol I bound to extended and displaced DNA section - closed conformation Type: COMPLEX / Entity ID: #1-#4 / Source: RECOMBINANT |
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Molecular weight | Value: 75 kDa/nm / Experimental value: NO |
Source (natural) | Organism: ![]() ![]() |
Source (recombinant) | Organism: ![]() ![]() |
Buffer solution | pH: 8.5 |
Specimen | Embedding applied: YES / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES |
Specimen support | Grid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R0.6/1 |
EM embedding | Material: water |
Vitrification | Cryogen name: ETHANE |
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Electron microscopy imaging
Experimental equipment | ![]() Model: Titan Krios / Image courtesy: FEI Company |
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Microscopy | Model: FEI TITAN KRIOS |
Electron gun | Electron source: ![]() |
Electron lens | Mode: BRIGHT FIELD / Nominal defocus max: 2500 nm / Nominal defocus min: 1000 nm |
Image recording | Electron dose: 45 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) |
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Processing
CTF correction | Type: PHASE FLIPPING AND AMPLITUDE CORRECTION | ||||||||||||||||||||||||
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3D reconstruction | Resolution: 4.3 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 65904 / Symmetry type: POINT | ||||||||||||||||||||||||
Refinement | Cross valid method: NONE Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2 | ||||||||||||||||||||||||
Displacement parameters | Biso mean: 145.53 Å2 | ||||||||||||||||||||||||
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