[English] 日本語
Yorodumi- PDB-8umu: Atomic model of the human CTF18-RFC-PCNA binary complex in the fo... -
+Open data
-Basic information
Entry | Database: PDB / ID: 8umu | ||||||
---|---|---|---|---|---|---|---|
Title | Atomic model of the human CTF18-RFC-PCNA binary complex in the four-subunit binding state (state 3) | ||||||
Components |
| ||||||
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 / Elg1 RFC-like complex / DNA replication factor C complex / Ctf18 RFC-like complex / response to organophosphorus / mitotic telomere maintenance via semi-conservative replication / DNA clamp loader activity / purine-specific mismatch base pair DNA N-glycosylase activity ...positive regulation of deoxyribonuclease activity / dinucleotide insertion or deletion binding / PCNA-p21 complex / Elg1 RFC-like complex / DNA replication factor C complex / Ctf18 RFC-like complex / response to organophosphorus / mitotic telomere maintenance via semi-conservative replication / DNA clamp loader activity / purine-specific mismatch base pair DNA N-glycosylase activity / positive regulation of DNA-directed DNA polymerase activity / nuclear lamina / MutLalpha complex binding / 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) / Mismatch repair (MMR) directed by MSH2:MSH6 (MutSalpha) / Transcription of E2F targets under negative control by DREAM complex / Polymerase switching on the C-strand of the telomere / Removal of the Flap Intermediate from the C-strand / replisome / DNA strand elongation involved in DNA replication / response to L-glutamate / DNA duplex unwinding / HDR through Single Strand Annealing (SSA) / histone acetyltransferase binding / Impaired BRCA2 binding to RAD51 / leading strand elongation / DNA synthesis involved in DNA repair / DNA polymerase processivity factor activity / G1/S-Specific Transcription / replication fork processing / response to dexamethasone / nuclear replication fork / Presynaptic phase of homologous DNA pairing and strand exchange / SUMOylation of DNA replication proteins / estrous cycle / PCNA-Dependent Long Patch Base Excision Repair / cyclin-dependent protein kinase holoenzyme complex / mismatch repair / translesion synthesis / ATP-dependent activity, acting on DNA / response to cadmium ion / Activation of ATR in response to replication stress / DNA polymerase binding / base-excision repair, gap-filling / epithelial cell differentiation / positive regulation of DNA repair / Translesion synthesis by REV1 / Translesion synthesis by POLK / Gap-filling DNA repair synthesis and ligation in GG-NER / Translesion synthesis by POLI / TP53 Regulates Transcription of Genes Involved in G2 Cell Cycle Arrest / replication fork / positive regulation of DNA replication / male germ cell nucleus / liver regeneration / nuclear estrogen receptor binding / Recognition of DNA damage by PCNA-containing replication complex / Termination of translesion DNA synthesis / Translesion Synthesis by POLH / HDR through Homologous Recombination (HRR) / G2/M DNA damage checkpoint / 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 UV / cellular response to xenobiotic stimulus / response to estradiol / E3 ubiquitin ligases ubiquitinate target proteins / heart development / Processing of DNA double-strand break ends / Regulation of TP53 Activity through Phosphorylation / DNA replication / chromosome, telomeric region / damaged DNA binding / nuclear body / cell cycle / DNA repair / centrosome / chromatin binding / chromatin / protein-containing complex binding / negative regulation of transcription by RNA polymerase II / enzyme binding / ATP hydrolysis activity / DNA binding / extracellular exosome / nucleoplasm / ATP binding / membrane / identical protein binding / nucleus Similarity search - Function | ||||||
Biological species | Homo sapiens (human) | ||||||
Method | ELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.16 Å | ||||||
Authors | Wang, F. / He, Q. / Li, H. | ||||||
Funding support | United States, 1items
| ||||||
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 |
|
-Structure visualization
Structure viewer | Molecule: MolmilJmol/JSmol |
---|
-Downloads & links
-Download
PDBx/mmCIF format | 8umu.cif.gz | 526.4 KB | Display | PDBx/mmCIF format |
---|---|---|---|---|
PDB format | pdb8umu.ent.gz | 415.6 KB | Display | PDB format |
PDBx/mmJSON format | 8umu.json.gz | Tree view | PDBx/mmJSON format | |
Others | Other downloads |
-Validation report
Arichive directory | https://data.pdbj.org/pub/pdb/validation_reports/um/8umu ftp://data.pdbj.org/pub/pdb/validation_reports/um/8umu | HTTPS FTP |
---|
-Related structure data
Related structure data | 42384MC 8umtC 8umvC 8umwC 8umyC 8un0C 8unjC M: map data used to model this data C: citing same article (ref.) |
---|---|
Similar structure data | Similarity search - Function & homologyF&H Search |
-Links
-Assembly
Deposited unit |
|
---|---|
1 |
|
-Components
-Protein , 2 types, 4 molecules AFGH
#1: Protein | Mass: 108520.977 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 |
---|---|
#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 |
---|---|
#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 |
-Non-polymers , 3 types, 7 molecules
#7: Chemical | #8: Chemical | #9: Chemical | ChemComp-ADP / | |
---|
-Details
Has ligand of interest | Y |
---|
-Experimental details
-Experiment
Experiment | Method: ELECTRON MICROSCOPY |
---|---|
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-#6 / Source: RECOMBINANT |
---|---|
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 |
Specimen support | Grid type: Quantifoil R2/1 |
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 |
---|---|
Microscopy | Model: FEI TITAN KRIOS |
Electron gun | Electron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM |
Electron lens | Mode: BRIGHT FIELDBright-field microscopy / 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 | ||||||||||||||||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
3D reconstruction | Resolution: 3.16 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 183349 / Symmetry type: POINT | ||||||||||||||||||||||||
Atomic model building | B value: 123 / Protocol: RIGID BODY FIT / Space: REAL | ||||||||||||||||||||||||
Refine LS restraints |
|