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Yorodumi- PDB-8un0: Atomic model of the human CTF18-RFC-PCNA-DNA ternary complex with... -
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-Basic information
Entry | Database: PDB / ID: 8un0 | ||||||
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Title | Atomic model of the human CTF18-RFC-PCNA-DNA ternary complex with cracked and closed PCNA (state 7) | ||||||
Components |
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Keywords | REPLICATION/DNA / DNA clamp loader complex / REPLICATION-DNA complex | ||||||
Function / homology | Function and homology information positive regulation of deoxyribonuclease activity / dinucleotide insertion or deletion binding / PCNA-p21 complex / response to organophosphorus / Ctf18 RFC-like complex / DNA clamp loader activity / DNA replication factor C complex / Elg1 RFC-like complex / mitotic telomere maintenance via semi-conservative replication / purine-specific mismatch base pair DNA N-glycosylase activity ...positive regulation of deoxyribonuclease activity / dinucleotide insertion or deletion binding / PCNA-p21 complex / response to organophosphorus / Ctf18 RFC-like complex / DNA clamp loader activity / DNA replication factor C complex / Elg1 RFC-like complex / mitotic telomere maintenance via semi-conservative replication / purine-specific mismatch base pair DNA N-glycosylase activity / nuclear lamina / MutLalpha complex binding / positive regulation of DNA-directed DNA polymerase activity / Polymerase switching / Telomere C-strand (Lagging Strand) Synthesis / Processive synthesis on the lagging strand / PCNA complex / Removal of the Flap Intermediate / Processive synthesis on the C-strand of the telomere / Mismatch repair (MMR) directed by MSH2:MSH3 (MutSbeta) / Polymerase switching on the C-strand of the telomere / Mismatch repair (MMR) directed by MSH2:MSH6 (MutSalpha) / Transcription of E2F targets under negative control by DREAM complex / Removal of the Flap Intermediate from the C-strand / replisome / Activation of ATR in response to replication stress / DNA duplex unwinding / response to L-glutamate / HDR through Single Strand Annealing (SSA) / Impaired BRCA2 binding to RAD51 / DNA strand elongation involved in DNA replication / histone acetyltransferase binding / DNA synthesis involved in DNA repair / DNA polymerase processivity factor activity / positive regulation of DNA replication / G1/S-Specific Transcription / replication fork processing / response to dexamethasone / leading strand elongation / nuclear replication fork / cyclin-dependent protein kinase holoenzyme complex / Presynaptic phase of homologous DNA pairing and strand exchange / SUMOylation of DNA replication proteins / estrous cycle / epithelial cell differentiation / PCNA-Dependent Long Patch Base Excision Repair / positive regulation of DNA repair / ATP-dependent activity, acting on DNA / mismatch repair / male germ cell nucleus / translesion synthesis / response to cadmium ion / DNA polymerase binding / liver regeneration / Translesion synthesis by REV1 / Translesion synthesis by POLK / base-excision repair, gap-filling / Translesion synthesis by POLI / Gap-filling DNA repair synthesis and ligation in GG-NER / TP53 Regulates Transcription of Genes Involved in G2 Cell Cycle Arrest / replication fork / nuclear estrogen receptor binding / Recognition of DNA damage by PCNA-containing replication complex / Termination of translesion DNA synthesis / G2/M DNA damage checkpoint / Translesion Synthesis by POLH / HDR through Homologous Recombination (HRR) / Dual Incision in GG-NER / DNA-templated DNA replication / receptor tyrosine kinase binding / cellular response to hydrogen peroxide / Dual incision in TC-NER / Gap-filling DNA repair synthesis and ligation in TC-NER / cellular response to xenobiotic stimulus / cellular response to UV / Processing of DNA double-strand break ends / E3 ubiquitin ligases ubiquitinate target proteins / response to estradiol / heart development / chromosome, telomeric region / DNA replication / Regulation of TP53 Activity through Phosphorylation / damaged DNA binding / nuclear body / DNA repair / chromatin binding / centrosome / chromatin / protein-containing complex binding / enzyme binding / negative regulation of transcription by RNA polymerase II / ATP hydrolysis activity / DNA binding / extracellular exosome / nucleoplasm / ATP binding / identical protein binding / membrane / nucleus / cytosol Similarity search - Function | ||||||
Biological species | Homo sapiens (human) synthetic construct (others) | ||||||
Method | ELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3 Å | ||||||
Authors | Wang, F. / He, Q. / Li, H. | ||||||
Funding support | United States, 1items
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Citation | Journal: Proc Natl Acad Sci U S A / Year: 2024 Title: Cryo-EM reveals a nearly complete PCNA loading process and unique features of the human alternative clamp loader CTF18-RFC. Authors: Qing He / Feng Wang / Michael E O'Donnell / Huilin Li / Abstract: The DNA sliding clamp PCNA is a multipurpose platform for DNA polymerases and many other proteins involved in DNA metabolism. The topologically closed PCNA ring needs to be cracked open and loaded ...The DNA sliding clamp PCNA is a multipurpose platform for DNA polymerases and many other proteins involved in DNA metabolism. The topologically closed PCNA ring needs to be cracked open and loaded onto DNA by a clamp loader, e.g., the well-studied pentameric ATPase complex RFC (RFC1-5). The CTF18-RFC complex is an alternative clamp loader found recently to bind the leading strand DNA polymerase ε and load PCNA onto leading strand DNA, but its structure and the loading mechanism have been unknown. By cryo-EM analysis of in vitro assembled human CTF18-RFC-DNA-PCNA complex, we have captured seven loading intermediates, revealing a detailed PCNA loading mechanism onto a 3'-ss/dsDNA junction by CTF18-RFC. Interestingly, the alternative loader has evolved a highly mobile CTF18 AAA+ module likely to lower the loading activity, perhaps to avoid competition with the RFC and to limit its role to leading strand clamp loading. To compensate for the lost stability due to the mobile AAA+ module, CTF18 has evolved a unique β-hairpin motif that reaches across RFC2 to interact with RFC5, thereby stabilizing the pentameric complex. Further, we found that CTF18 also contains a separation pin to locally melt DNA from the 3'-end of the primer; this ensures its ability to load PCNA to any 3'-ss/dsDNA junction, facilitated by the binding energy of the E-plug to the major groove. Our study reveals unique structural features of the human CTF18-RFC and contributes to a broader understanding of PCNA loading by the alternative clamp loaders. | ||||||
History |
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-Structure visualization
Structure viewer | Molecule: MolmilJmol/JSmol |
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-Downloads & links
-Download
PDBx/mmCIF format | 8un0.cif.gz | 560.2 KB | Display | PDBx/mmCIF format |
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PDB format | pdb8un0.ent.gz | 442.3 KB | Display | PDB format |
PDBx/mmJSON format | 8un0.json.gz | Tree view | PDBx/mmJSON format | |
Others | Other downloads |
-Validation report
Summary document | 8un0_validation.pdf.gz | 1.7 MB | Display | wwPDB validaton report |
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Full document | 8un0_full_validation.pdf.gz | 1.7 MB | Display | |
Data in XML | 8un0_validation.xml.gz | 83.3 KB | Display | |
Data in CIF | 8un0_validation.cif.gz | 124.7 KB | Display | |
Arichive directory | https://data.pdbj.org/pub/pdb/validation_reports/un/8un0 ftp://data.pdbj.org/pub/pdb/validation_reports/un/8un0 | HTTPS FTP |
-Related structure data
Related structure data | 42389MC 8umtC 8umuC 8umvC 8umwC 8umyC 8unjC M: map data used to model this data C: citing same article (ref.) |
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Similar structure data | Similarity search - Function & homologyF&H Search |
-Links
-Assembly
Deposited unit |
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1 |
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-Components
-Protein , 2 types, 4 molecules AFGH
#1: Protein | Mass: 107523.984 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Homo sapiens (human) / Gene: CHTF18 Production host: Insect cell expression vector pTIE1 (others) References: UniProt: Q8WVB6 |
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#6: Protein | Mass: 28795.752 Da / Num. of mol.: 3 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Homo sapiens (human) / Gene: PCNA Production host: Insect cell expression vector pTIE1 (others) References: UniProt: P12004 |
-Replication factor C subunit ... , 4 types, 4 molecules BCDE
#2: Protein | Mass: 39203.207 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Homo sapiens (human) / Gene: RFC2 Production host: Insect cell expression vector pTIE1 (others) References: UniProt: P35250 |
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#3: Protein | Mass: 38545.512 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Homo sapiens (human) / Gene: RFC5 Production host: Insect cell expression vector pTIE1 (others) References: UniProt: P40937 |
#4: Protein | Mass: 39735.711 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Homo sapiens (human) / Gene: RFC4 Production host: Insect cell expression vector pTIE1 (others) References: UniProt: P35249 |
#5: Protein | Mass: 40614.332 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Homo sapiens (human) / Gene: RFC3 Production host: Insect cell expression vector pTIE1 (others) References: UniProt: P40938 |
-DNA chain , 2 types, 2 molecules IJ
#7: DNA chain | Mass: 12214.818 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others) |
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#8: DNA chain | Mass: 6136.008 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others) |
-Non-polymers , 3 types, 9 molecules
#9: Chemical | ChemComp-MG / #10: Chemical | ChemComp-AGS / #11: Chemical | ChemComp-ADP / | |
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-Details
Has ligand of interest | Y |
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-Experimental details
-Experiment
Experiment | Method: ELECTRON MICROSCOPY |
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EM experiment | Aggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction |
-Sample preparation
Component | Name: the human clamp-clamp loader complex PCNA-CTF18 / Type: COMPLEX / Entity ID: #1-#8 / Source: RECOMBINANT |
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Molecular weight | Value: 0.3 MDa / Experimental value: YES |
Source (natural) | Organism: Homo sapiens (human) |
Source (recombinant) | Organism: Insect cell expression vector pTIE1 (others) |
Buffer solution | pH: 7.5 |
Specimen | Conc.: 0.3 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES |
Vitrification | Instrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 280 K |
-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: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM |
Electron lens | Mode: BRIGHT FIELD / Nominal defocus max: 1900 nm / Nominal defocus min: 1200 nm |
Image recording | Electron dose: 60 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) |
-Processing
CTF correction | Type: PHASE FLIPPING AND AMPLITUDE CORRECTION |
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3D reconstruction | Resolution: 3 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 75789 / Symmetry type: POINT |
Atomic model building | B value: 123 / Protocol: RIGID BODY FIT / Space: REAL |