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- PDB-8dqz: Intermediate state of RFC:PCNA bound to a 3' ss/dsDNA junction -

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Basic information

Entry
Database: PDB / ID: 8dqz
TitleIntermediate state of RFC:PCNA bound to a 3' ss/dsDNA junction
Components
  • (Replication factor C subunit ...) x 5
  • DNA (5'-D(P*CP*CP*CP*CP*GP*GP*GP*GP*CP*CP*CP*CP*CP*CP*CP*GP*GP*C)-3')
  • DNA (5'-D(P*TP*TP*TP*TP*TP*TP*CP*GP*GP*GP*GP*GP*GP*GP*CP*CP*CP*CP*GP*GP*GP*G)-3')
  • Proliferating cell nuclear antigen
KeywordsREPLICATION/DNA / REPLICATION-DNA complex
Function / homology
Function and homology information


DNA clamp unloader activity / DNA clamp unloading / Mismatch repair (MMR) directed by MSH2:MSH6 (MutSalpha) / Gap-filling DNA repair synthesis and ligation in GG-NER / meiotic mismatch repair / Processive synthesis on the lagging strand / Removal of the Flap Intermediate / Ctf18 RFC-like complex / Rad17 RFC-like complex / DNA replication factor C complex ...DNA clamp unloader activity / DNA clamp unloading / Mismatch repair (MMR) directed by MSH2:MSH6 (MutSalpha) / Gap-filling DNA repair synthesis and ligation in GG-NER / meiotic mismatch repair / Processive synthesis on the lagging strand / Removal of the Flap Intermediate / Ctf18 RFC-like complex / Rad17 RFC-like complex / 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 / DNA clamp loader activity / Translesion Synthesis by POLH / Translesion synthesis by REV1 / Translesion synthesis by POLK / Translesion synthesis by POLI / establishment of mitotic sister chromatid cohesion / PCNA complex / DNA replication checkpoint signaling / 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 / DNA damage checkpoint signaling / replication fork / positive regulation of DNA replication / 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
Replication factor C subunit 1 / DNA replication factor RFC1, C-terminal / Replication factor RFC1 C terminal 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 ...Replication factor C subunit 1 / DNA replication factor RFC1, C-terminal / Replication factor RFC1 C terminal 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 / : / BRCA1 C Terminus (BRCT) domain / breast cancer carboxy-terminal domain / BRCT domain profile. / BRCT domain / BRCT domain superfamily / 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
PHOSPHOTHIOPHOSPHORIC ACID-ADENYLATE ESTER / GUANOSINE-5'-DIPHOSPHATE / DNA / DNA (> 10) / Proliferating cell nuclear antigen / Replication factor C subunit 5 / Replication factor C subunit 3 / Replication factor C subunit 1 / Replication factor C subunit 4 / Replication factor C subunit 2
Similarity search - Component
Biological speciesSaccharomyces cerevisiae (brewer's yeast)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.92 Å
AuthorsSchrecker, M. / Hite, R.K.
Funding support United States, 3items
OrganizationGrant numberCountry
National Institutes of Health/National Cancer Institute (NIH/NCI)CA008748 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM107239 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM127428 United States
CitationJournal: Elife / Year: 2022
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.
History
DepositionJul 20, 2022Deposition site: RCSB / Processing site: RCSB
Revision 1.0Aug 24, 2022Provider: repository / Type: Initial release
Revision 1.1Feb 14, 2024Group: Data collection / Category: chem_comp_atom / chem_comp_bond

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Replication factor C subunit 1
B: Replication factor C subunit 4
C: Replication factor C subunit 3
D: Replication factor C subunit 2
E: Replication factor C subunit 5
F: Proliferating cell nuclear antigen
G: Proliferating cell nuclear antigen
H: Proliferating cell nuclear antigen
I: DNA (5'-D(P*TP*TP*TP*TP*TP*TP*CP*GP*GP*GP*GP*GP*GP*GP*CP*CP*CP*CP*GP*GP*GP*G)-3')
J: DNA (5'-D(P*CP*CP*CP*CP*GP*GP*GP*GP*CP*CP*CP*CP*CP*CP*CP*GP*GP*C)-3')
hetero molecules


Theoretical massNumber of molelcules
Total (without water)363,78819
Polymers361,15410
Non-polymers2,6339
Water00
1


  • Idetical with deposited unit
  • defined by author
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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Replication factor C subunit ... , 5 types, 5 molecules ABCDE

#1: Protein Replication factor C subunit 1 / Replication factor C1 / Activator 1 95 kDa subunit / Cell division control protein 44


Mass: 101689.086 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (brewer's yeast)
Gene: RFC1, CDC44, YOR217W, YOR50-7 / Production host: Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: P38630
#2: Protein Replication factor C subunit 4 / Replication factor C4 / Activator 1 37 kDa subunit


Mass: 36201.039 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (brewer's yeast)
Gene: RFC4, YOL094C, O0923 / Production host: Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: P40339
#3: Protein Replication factor C subunit 3 / Replication factor C3 / Activator 1 40 kDa subunit


Mass: 38254.543 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (brewer's yeast)
Gene: RFC3, YNL290W, N0533 / Production host: Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: P38629
#4: Protein Replication factor C subunit 2 / Replication factor C2 / Activator 1 41 kDa subunit


Mass: 39794.473 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (brewer's yeast)
Gene: RFC2, YJR068W, J1808 / Production host: Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: P40348
#5: Protein Replication factor C subunit 5 / Replication factor C5 / Activator 1 40 kDa subunit


Mass: 39993.582 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (brewer's yeast)
Gene: RFC5, YBR087W, YBR0810 / Production host: Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: P38251

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Protein , 1 types, 3 molecules FGH

#6: Protein Proliferating cell nuclear antigen / PCNA


Mass: 30991.285 Da / Num. of mol.: 3
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (brewer's yeast)
Strain: ATCC 204508 / S288c / Gene: POL30, YBR088C, YBR0811 / Production host: Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: P15873

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DNA chain , 2 types, 2 molecules IJ

#7: DNA chain DNA (5'-D(P*TP*TP*TP*TP*TP*TP*CP*GP*GP*GP*GP*GP*GP*GP*CP*CP*CP*CP*GP*GP*GP*G)-3')


Mass: 6847.372 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Saccharomyces cerevisiae (brewer's yeast)
#8: DNA chain DNA (5'-D(P*CP*CP*CP*CP*GP*GP*GP*GP*CP*CP*CP*CP*CP*CP*CP*GP*GP*C)-3')


Mass: 5400.458 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Saccharomyces cerevisiae (brewer's yeast)

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Non-polymers , 3 types, 9 molecules

#9: 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
#10: Chemical
ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: Mg / Feature type: SUBJECT OF INVESTIGATION
#11: Chemical ChemComp-GDP / GUANOSINE-5'-DIPHOSPHATE


Type: RNA linking / Mass: 443.201 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C10H15N5O11P2 / Feature type: SUBJECT OF INVESTIGATION / Comment: GDP, 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: Intermediate state of RFC:PCNA bound to a 3' ss/dsDNA junction
Type: COMPLEX / Entity ID: #1-#8 / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)
Source (recombinant)Organism: Saccharomyces cerevisiae (brewer's yeast)
Buffer solutionpH: 7.6
Buffer component
IDConc.NameBuffer-ID
125 mMHEPES-KOH1
2300 mMpotassium acetate1
37 mMmagnesium acetate1
SpecimenConc.: 1 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GRAPHENE OXIDE / Grid mesh size: 400 divisions/in. / Grid type: Quantifoil
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 297 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: 2000 nm / Nominal defocus min: 700 nm
Image recordingElectron dose: 66 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

SoftwareName: PHENIX / Version: 1.20.1_4487: / Classification: refinement
EM software
IDNameVersionCategory
2SerialEMimage acquisition
7PHENIXmodel fitting
12cryoSPARC3.33D reconstruction
13PHENIXmodel refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 2.92 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 41190 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00221880
ELECTRON MICROSCOPYf_angle_d0.49229759
ELECTRON MICROSCOPYf_dihedral_angle_d12.5983249
ELECTRON MICROSCOPYf_chiral_restr0.0383457
ELECTRON MICROSCOPYf_plane_restr0.0033639

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