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- PDB-8umv: Atomic model of the human CTF18-RFC-PCNA-DNA ternary complex with... -

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Basic information

Entry
Database: PDB / ID: 8umv
TitleAtomic model of the human CTF18-RFC-PCNA-DNA ternary complex with narrow PCNA opening state I (state 5)
Components
  • (Replication factor C subunit ...) x 4
  • Chromosome transmission fidelity protein 18 homolog
  • DNA (20-MER)
  • DNA (40-MER)
  • Proliferating cell nuclear antigen
KeywordsREPLICATION/DNA / DNA clamp loader complex / REPLICATION-DNA complex
Function / homology
Function and homology information


response to organophosphorus / Elg1 RFC-like complex / DNA replication factor C complex / Ctf18 RFC-like complex / positive regulation of deoxyribonuclease activity / dinucleotide insertion or deletion binding / PCNA-p21 complex / mitotic telomere maintenance via semi-conservative replication / purine-specific mismatch base pair DNA N-glycosylase activity / MutLalpha complex binding ...response to organophosphorus / Elg1 RFC-like complex / DNA replication factor C complex / Ctf18 RFC-like complex / positive regulation of deoxyribonuclease activity / dinucleotide insertion or deletion binding / PCNA-p21 complex / mitotic telomere maintenance via semi-conservative replication / purine-specific mismatch base pair DNA N-glycosylase activity / MutLalpha complex binding / positive regulation of DNA-directed DNA polymerase activity / nuclear lamina / 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 / Polymerase switching on the C-strand of the telomere / Mismatch repair (MMR) directed by MSH2:MSH3 (MutSbeta) / Mismatch repair (MMR) directed by MSH2:MSH6 (MutSalpha) / Removal of the Flap Intermediate from the C-strand / Transcription of E2F targets under negative control by DREAM complex / replisome / : / HDR through Single Strand Annealing (SSA) / response to L-glutamate / Impaired BRCA2 binding to RAD51 / histone acetyltransferase binding / DNA synthesis involved in DNA repair / DNA strand elongation involved in DNA replication / DNA polymerase processivity factor activity / G1/S-Specific Transcription / leading strand elongation / replication fork processing / response to dexamethasone / nuclear replication fork / Presynaptic phase of homologous DNA pairing and strand exchange / SUMOylation of DNA replication proteins / ATP-dependent activity, acting on DNA / PCNA-Dependent Long Patch Base Excision Repair / mismatch repair / Activation of ATR in response to replication stress / translesion synthesis / response to cadmium ion / cyclin-dependent protein kinase holoenzyme complex / estrous cycle / DNA polymerase binding / base-excision repair, gap-filling / epithelial cell differentiation / positive regulation of DNA repair / TP53 Regulates Transcription of Genes Involved in G2 Cell Cycle Arrest / Translesion synthesis by REV1 / Translesion synthesis by POLK / liver regeneration / Translesion synthesis by POLI / Gap-filling DNA repair synthesis and ligation in GG-NER / positive regulation of DNA replication / replication fork / male germ cell nucleus / nuclear estrogen receptor binding / Termination of translesion DNA synthesis / Recognition of DNA damage by PCNA-containing replication complex / Translesion Synthesis by POLH / G2/M DNA damage checkpoint / HDR through Homologous Recombination (HRR) / receptor tyrosine kinase binding / Dual Incision in GG-NER / cellular response to hydrogen peroxide / DNA-templated DNA replication / 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 clamp loader activity / damaged DNA binding / DNA replication / chromosome, telomeric region / nuclear body / DNA repair / centrosome / chromatin binding / protein-containing complex binding / chromatin / enzyme binding / negative regulation of transcription by RNA polymerase II / ATP hydrolysis activity / DNA binding / extracellular exosome / nucleoplasm / ATP binding / identical protein binding / nucleus / membrane / cytosol
Similarity search - Function
: / Replication factor C subunit 3, C-terminal domain / RCF1/5-like, AAA+ ATPase lid domain / Replication factor C, C-terminal / Replication factor C C-terminal domain / : / DNA polymerase III, delta subunit / : / DNA polymerase III, clamp loader complex, gamma/delta/delta subunit, C-terminal / Proliferating cell nuclear antigen signature 2. ...: / Replication factor C subunit 3, C-terminal domain / RCF1/5-like, AAA+ ATPase lid domain / Replication factor C, C-terminal / Replication factor C C-terminal domain / : / DNA polymerase III, delta subunit / : / DNA polymerase III, clamp loader complex, gamma/delta/delta subunit, C-terminal / Proliferating cell nuclear antigen signature 2. / Proliferating cell nuclear antigen, PCNA, conserved site / Proliferating cell nuclear antigen signature 1. / Proliferating cell nuclear antigen, PCNA / Proliferating cell nuclear antigen, PCNA, N-terminal / Proliferating cell nuclear antigen, PCNA, C-terminal / Proliferating cell nuclear antigen, N-terminal domain / Proliferating cell nuclear antigen, C-terminal domain / : / ATPase family associated with various cellular activities (AAA) / ATPase, AAA-type, core / ATPases associated with a variety of cellular activities / AAA+ ATPase domain / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
ADENOSINE-5'-DIPHOSPHATE / PHOSPHOTHIOPHOSPHORIC ACID-ADENYLATE ESTER / DNA / DNA (> 10) / Proliferating cell nuclear antigen / Replication factor C subunit 4 / Replication factor C subunit 2 / Replication factor C subunit 5 / Replication factor C subunit 3 / Chromosome transmission fidelity protein 18 homolog
Similarity search - Component
Biological speciesHomo sapiens (human)
synthetic construct (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.75 Å
AuthorsWang, F. / He, Q. / Li, H.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM131754 United States
CitationJournal: 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
DepositionOct 18, 2023Deposition site: RCSB / Processing site: RCSB
Revision 1.0May 8, 2024Provider: repository / Type: Initial release
Revision 1.1May 15, 2024Group: Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_PubMed / _citation.title

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Chromosome transmission fidelity protein 18 homolog
B: Replication factor C subunit 2
C: Replication factor C subunit 5
D: Replication factor C subunit 4
E: Replication factor C subunit 3
F: Proliferating cell nuclear antigen
G: Proliferating cell nuclear antigen
H: Proliferating cell nuclear antigen
I: DNA (40-MER)
J: DNA (20-MER)
hetero molecules


Theoretical massNumber of molelcules
Total (without water)372,97819
Polymers370,36110
Non-polymers2,6179
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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Protein , 2 types, 4 molecules AFGH

#1: Protein Chromosome transmission fidelity protein 18 homolog


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
#6: Protein Proliferating cell nuclear antigen / PCNA / Cyclin


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: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
References: UniProt: P12004

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Replication factor C subunit ... , 4 types, 4 molecules BCDE

#2: Protein Replication factor C subunit 2 / Activator 1 40 kDa subunit / A1 40 kDa subunit / Activator 1 subunit 2 / Replication factor C 40 ...Activator 1 40 kDa subunit / A1 40 kDa subunit / Activator 1 subunit 2 / Replication factor C 40 kDa subunit / RFC40


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 Replication factor C subunit 5 / Activator 1 36 kDa subunit / A1 36 kDa subunit / Activator 1 subunit 5 / Replication factor C 36 ...Activator 1 36 kDa subunit / A1 36 kDa subunit / Activator 1 subunit 5 / Replication factor C 36 kDa subunit / RFC36


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 Replication factor C subunit 4


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 Replication factor C subunit 3 / Activator 1 38 kDa subunit / A1 38 kDa subunit / Activator 1 subunit 3 / Replication factor C 38 ...Activator 1 38 kDa subunit / A1 38 kDa subunit / Activator 1 subunit 3 / Replication factor C 38 kDa subunit / RFC38


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

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DNA chain , 2 types, 2 molecules IJ

#7: DNA chain DNA (40-MER)


Mass: 12214.818 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#8: DNA chain DNA (20-MER)


Mass: 6136.008 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)

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Non-polymers , 3 types, 9 molecules

#9: Chemical
ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: Mg / Feature type: SUBJECT OF INVESTIGATION
#10: Chemical
ChemComp-AGS / PHOSPHOTHIOPHOSPHORIC ACID-ADENYLATE ESTER / ATP-GAMMA-S / ADENOSINE 5'-(3-THIOTRIPHOSPHATE) / ADENOSINE 5'-(GAMMA-THIOTRIPHOSPHATE) / ADENOSINE-5'-DIPHOSPHATE MONOTHIOPHOSPHATE


Mass: 523.247 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: C10H16N5O12P3S / Feature type: SUBJECT OF INVESTIGATION / Comment: ATP-gamma-S, energy-carrying molecule analogue*YM
#11: Chemical ChemComp-ADP / ADENOSINE-5'-DIPHOSPHATE


Mass: 427.201 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C10H15N5O10P2 / Feature type: SUBJECT OF INVESTIGATION / Comment: ADP, energy-carrying molecule*YM

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Details

Has ligand of interestY

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: the human clamp-clamp loader complex PCNA-CTF18 / Type: COMPLEX / Entity ID: #1-#6 / Source: RECOMBINANT
Molecular weightValue: 0.3 MDa / Experimental value: YES
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Insect cell expression vector pTIE1 (others)
Buffer solutionpH: 7.5
SpecimenConc.: 0.3 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid type: Quantifoil R2/1
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 280 K

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 1900 nm / Nominal defocus min: 1200 nm
Image recordingElectron dose: 60 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 2.75 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 212091 / Symmetry type: POINT
Atomic model buildingB value: 123 / Protocol: RIGID BODY FIT / Space: REAL
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00322309
ELECTRON MICROSCOPYf_angle_d0.62530398
ELECTRON MICROSCOPYf_dihedral_angle_d15.5728509
ELECTRON MICROSCOPYf_chiral_restr0.0433536
ELECTRON MICROSCOPYf_plane_restr0.0063723

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