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Open data
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Basic information
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Title | Intermediate state of RFC:PCNA bound to a 3' ss/dsDNA junction | ||||||||||||
![]() | RFC-PCNA intermediate state | ||||||||||||
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![]() | REPLICATION-DNA complex | ||||||||||||
Function / homology | ![]() DNA clamp unloading / Mismatch repair (MMR) directed by MSH2:MSH6 (MutSalpha) / Gap-filling DNA repair synthesis and ligation in GG-NER / meiotic mismatch repair / Rad17 RFC-like complex / Processive synthesis on the lagging strand / Removal of the Flap Intermediate / Ctf18 RFC-like complex / DNA clamp loader activity / DNA replication factor C complex ...DNA clamp unloading / Mismatch repair (MMR) directed by MSH2:MSH6 (MutSalpha) / Gap-filling DNA repair synthesis and ligation in GG-NER / meiotic mismatch repair / Rad17 RFC-like complex / Processive synthesis on the lagging strand / Removal of the Flap Intermediate / Ctf18 RFC-like complex / DNA clamp loader activity / DNA replication factor C complex / Elg1 RFC-like complex / E3 ubiquitin ligases ubiquitinate target proteins / Polymerase switching / SUMOylation of DNA replication proteins / positive regulation of DNA metabolic process / maintenance of DNA trinucleotide repeats / Translesion Synthesis by POLH / Translesion synthesis by REV1 / Translesion synthesis by POLK / Translesion synthesis by POLI / establishment of mitotic sister chromatid cohesion / DNA replication checkpoint signaling / PCNA complex / Activation of ATR in response to replication stress / Termination of translesion DNA synthesis / lagging strand elongation / postreplication repair / sister chromatid cohesion / silent mating-type cassette heterochromatin formation / mitotic sister chromatid cohesion / error-free translesion synthesis / DNA polymerase processivity factor activity / leading strand elongation / Gap-filling DNA repair synthesis and ligation in TC-NER / Dual incision in TC-NER / subtelomeric heterochromatin formation / mismatch repair / translesion synthesis / positive regulation of DNA repair / positive regulation of DNA replication / DNA damage checkpoint signaling / replication fork / nucleotide-excision repair / DNA-templated DNA replication / mitotic cell cycle / chromosome, telomeric region / cell division / DNA repair / ATP hydrolysis activity / DNA binding / ATP binding / identical protein binding / nucleus / cytosol Similarity search - Function | ||||||||||||
Biological species | ![]() ![]() | ||||||||||||
Method | single particle reconstruction / cryo EM / Resolution: 2.92 Å | ||||||||||||
![]() | Schrecker M / Hite RK | ||||||||||||
Funding support | ![]()
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![]() | ![]() Title: Multistep loading of a DNA sliding clamp onto DNA by replication factor C. Authors: Marina Schrecker / Juan C Castaneda / Sujan Devbhandari / Charanya Kumar / Dirk Remus / Richard K Hite / ![]() Abstract: The DNA sliding clamp proliferating cell nuclear antigen (PCNA) is an essential co-factor for many eukaryotic DNA metabolic enzymes. PCNA is loaded around DNA by the ATP-dependent clamp loader ...The DNA sliding clamp proliferating cell nuclear antigen (PCNA) is an essential co-factor for many eukaryotic DNA metabolic enzymes. PCNA is loaded around DNA by the ATP-dependent clamp loader replication factor C (RFC), which acts at single-stranded (ss)/double-stranded DNA (dsDNA) junctions harboring a recessed 3' end (3' ss/dsDNA junctions) and at DNA nicks. To illuminate the loading mechanism we have investigated the structure of RFC:PCNA bound to ATPγS and 3' ss/dsDNA junctions or nicked DNA using cryogenic electron microscopy. Unexpectedly, we observe open and closed PCNA conformations in the RFC:PCNA:DNA complex, revealing that PCNA can adopt an open, planar conformation that allows direct insertion of dsDNA, and raising the question of whether PCNA ring closure is mechanistically coupled to ATP hydrolysis. By resolving multiple DNA-bound states of RFC:PCNA we observe that partial melting facilitates lateral insertion into the central channel formed by RFC:PCNA. We also resolve the Rfc1 N-terminal domain and demonstrate that its single BRCT domain participates in coordinating DNA prior to insertion into the central RFC channel, which promotes PCNA loading on the lagging strand of replication forks in vitro. Combined, our data suggest a comprehensive and fundamentally revised model for the RFC-catalyzed loading of PCNA onto DNA. | ||||||||||||
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Structure visualization
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Downloads & links
-EMDB archive
Map data | ![]() | 108.2 MB | ![]() | |
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Header (meta data) | ![]() ![]() | 29.8 KB 29.8 KB | Display Display | ![]() |
FSC (resolution estimation) | ![]() | 12.7 KB | Display | ![]() |
Images | ![]() | 183.1 KB | ||
Filedesc metadata | ![]() | 8.1 KB | ||
Others | ![]() ![]() ![]() | 676.6 MB 200.3 MB 200.3 MB | ||
Archive directory | ![]() ![]() | HTTPS FTP |
-Related structure data
Related structure data | ![]() 8dqzMC ![]() 8dqwC ![]() 8dqxC ![]() 8dr0C ![]() 8dr1C ![]() 8dr3C ![]() 8dr4C ![]() 8dr5C ![]() 8dr6C ![]() 8dr7C 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 | RFC-PCNA intermediate state | ||||||||||||||||||||
Voxel size | X=Y=Z: 0.826 Å | ||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||
Details | EMDB XML:
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-Supplemental data
-Additional map: Density modified and 1.5x sampled map
File | emd_27666_additional_1.map | ||||||||||||
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Annotation | Density modified and 1.5x sampled map | ||||||||||||
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Density Histograms |
-Half map: A
File | emd_27666_half_map_1.map | ||||||||||||
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Annotation | A | ||||||||||||
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-Half map: B
File | emd_27666_half_map_2.map | ||||||||||||
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Annotation | B | ||||||||||||
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Density Histograms |
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Sample components
+Entire : Intermediate state of RFC:PCNA bound to a 3' ss/dsDNA junction
+Supramolecule #1: Intermediate state of RFC:PCNA bound to a 3' ss/dsDNA junction
+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: Proliferating cell nuclear antigen
+Macromolecule #7: DNA (5'-D(P*TP*TP*TP*TP*TP*TP*CP*GP*GP*GP*GP*GP*GP*GP*CP*CP*CP*CP...
+Macromolecule #8: DNA (5'-D(P*CP*CP*CP*CP*GP*GP*GP*GP*CP*CP*CP*CP*CP*CP*CP*GP*GP*C)-3')
+Macromolecule #9: PHOSPHOTHIOPHOSPHORIC ACID-ADENYLATE ESTER
+Macromolecule #10: MAGNESIUM ION
+Macromolecule #11: GUANOSINE-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
Concentration | 1.0 mg/mL | ||||||||
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Buffer | pH: 7.6 Component:
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Grid | Model: Quantifoil / Material: GRAPHENE OXIDE / Mesh: 400 / Support film - Material: GRAPHENE OXIDE / Support film - topology: CONTINUOUS | ||||||||
Vitrification | Cryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 297 K / Instrument: FEI VITROBOT MARK IV |
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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: 66.0 e/Å2 |
Electron beam | Acceleration voltage: 300 kV / Electron source: ![]() |
Electron optics | Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.0 µm / Nominal defocus min: 0.7000000000000001 µm |
Experimental equipment | ![]() Model: Titan Krios / Image courtesy: FEI Company |