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- EMDB-25875: Cryo-EM 3D map of S. cerevisiae clamp loader RFC bound to two DNA... -
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Open data
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Basic information
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Title | Cryo-EM 3D map of S. cerevisiae clamp loader RFC bound to two DNA molecules, one at the 5'-recessed end and the other at the 3'-recessed end | ||||||||||||
![]() | 3D cryoEM map of yeast RFC-DNA1 complex | ||||||||||||
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![]() | DNA replication / DNA damage repair / RFC loader / PCNA clamp / DNA polymerase processivity factor / DNA binding protein-DNA complex | ||||||||||||
Function / homology | ![]() DNA clamp unloader activity / DNA clamp unloading / Gap-filling DNA repair synthesis and ligation in GG-NER / Ctf18 RFC-like complex / Rad17 RFC-like complex / DNA replication factor C complex / Elg1 RFC-like complex / Polymerase switching / DNA clamp loader activity / Translesion Synthesis by POLH ...DNA clamp unloader activity / DNA clamp unloading / Gap-filling DNA repair synthesis and ligation in GG-NER / Ctf18 RFC-like complex / Rad17 RFC-like complex / DNA replication factor C complex / Elg1 RFC-like complex / Polymerase switching / DNA clamp loader activity / Translesion Synthesis by POLH / Translesion synthesis by REV1 / Translesion synthesis by POLK / Translesion synthesis by POLI / DNA replication checkpoint signaling / Activation of ATR in response to replication stress / Termination of translesion DNA synthesis / sister chromatid cohesion / mitotic sister chromatid cohesion / leading strand elongation / Gap-filling DNA repair synthesis and ligation in TC-NER / Dual incision in TC-NER / mismatch repair / DNA damage checkpoint signaling / DNA-templated DNA replication / mitotic cell cycle / cell division / DNA repair / ATP hydrolysis activity / DNA binding / ATP binding / nucleus / cytosol Similarity search - Function | ||||||||||||
Biological species | synthetic construct (others) / ![]() ![]() | ||||||||||||
Method | single particle reconstruction / cryo EM / Resolution: 3.25 Å | ||||||||||||
![]() | Zheng F / Georgescu R | ||||||||||||
Funding support | ![]()
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![]() | ![]() Title: Cryo-EM structures reveal that RFC recognizes both the 3'- and 5'-DNA ends to load PCNA onto gaps for DNA repair. Authors: Fengwei Zheng / Roxana Georgescu / Nina Y Yao / Huilin Li / Michael E O'Donnell / ![]() Abstract: RFC uses ATP to assemble PCNA onto primed sites for replicative DNA polymerases δ and ε. The RFC pentamer forms a central chamber that binds 3' ss/ds DNA junctions to load PCNA onto DNA during ...RFC uses ATP to assemble PCNA onto primed sites for replicative DNA polymerases δ and ε. The RFC pentamer forms a central chamber that binds 3' ss/ds DNA junctions to load PCNA onto DNA during replication. We show here five structures that identify a second DNA binding site in RFC that binds a 5' duplex. This 5' DNA site is located between the N-terminal BRCT domain and AAA+ module of the large Rfc1 subunit. Our structures reveal ideal binding to a 7-nt gap, which includes 2 bp unwound by the clamp loader. Biochemical studies show enhanced binding to 5 and 10 nt gaps, consistent with the structural results. Because both 3' and 5' ends are present at a ssDNA gap, we propose that the 5' site facilitates RFC's PCNA loading activity at a DNA damage-induced gap to recruit gap-filling polymerases. These findings are consistent with genetic studies showing that base excision repair of gaps greater than 1 base requires PCNA and involves the 5' DNA binding domain of Rfc1. We further observe that a 5' end facilitates PCNA loading at an RPA coated 30-nt gap, suggesting a potential role of the RFC 5'-DNA site in lagging strand DNA synthesis. | ||||||||||||
History |
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Structure visualization
Supplemental images |
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Downloads & links
-EMDB archive
Map data | ![]() | 230.2 MB | ![]() | |
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Header (meta data) | ![]() ![]() | 21 KB 21 KB | Display Display | ![]() |
Images | ![]() | 45.5 KB | ||
Filedesc metadata | ![]() | 7.4 KB | ||
Archive directory | ![]() ![]() | HTTPS FTP |
-Validation report
Summary document | ![]() | 478.7 KB | Display | ![]() |
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Full document | ![]() | 478.2 KB | Display | |
Data in XML | ![]() | 7.1 KB | Display | |
Data in CIF | ![]() | 8.1 KB | Display | |
Arichive directory | ![]() ![]() | HTTPS FTP |
-Related structure data
Related structure data | ![]() 7tfkMC ![]() 7tfhC ![]() 7tfiC ![]() 7tfjC ![]() 7tflC C: citing same article ( M: atomic model generated by this map |
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Similar structure data | Similarity search - Function & homology ![]() |
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Links
EMDB pages | ![]() ![]() |
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Related items in Molecule of the Month |
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Map
File | ![]() | ||||||||||||||||||||
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Annotation | 3D cryoEM map of yeast RFC-DNA1 complex | ||||||||||||||||||||
Voxel size | X=Y=Z: 0.828 Å | ||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||
Details | EMDB XML:
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-Supplemental data
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Sample components
+Entire : RFC-PCNA-DNA1-DNA2
+Supramolecule #1: RFC-PCNA-DNA1-DNA2
+Supramolecule #2: dsDNA
+Supramolecule #3: Proteins
+Macromolecule #1: Replication factor C subunit 1
+Macromolecule #2: Replication factor C subunit 4
+Macromolecule #3: Replication factor C subunit 3
+Macromolecule #4: Replication factor C subunit 2
+Macromolecule #5: Replication factor C subunit 5
+Macromolecule #6: Template strand
+Macromolecule #7: Primer strand
+Macromolecule #8: PHOSPHOTHIOPHOSPHORIC ACID-ADENYLATE ESTER
+Macromolecule #9: MAGNESIUM ION
+Macromolecule #10: ADENOSINE-5'-DIPHOSPHATE
-Experimental details
-Structure determination
Method | cryo EM |
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![]() | single particle reconstruction |
Aggregation state | particle |
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Sample preparation
Buffer | pH: 7.5 |
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Vitrification | Cryogen name: ETHANE |
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Electron microscopy
Microscope | FEI TITAN KRIOS |
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Image recording | Film or detector model: GATAN K3 (6k x 4k) / Average electron dose: 65.0 e/Å2 |
Electron beam | Acceleration voltage: 300 kV / Electron source: ![]() |
Electron optics | Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 1.9000000000000001 µm / Nominal defocus min: 1.3 µm |
Experimental equipment | ![]() Model: Titan Krios / Image courtesy: FEI Company |
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Image processing
Startup model | Type of model: PDB ENTRY PDB model - PDB ID: |
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Final reconstruction | Resolution.type: BY AUTHOR / Resolution: 3.25 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 315619 |
Initial angle assignment | Type: NOT APPLICABLE |
Final angle assignment | Type: NOT APPLICABLE |