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- PDB-8w7s: Yeast replisome in state IV -

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Basic information

Entry
Database: PDB / ID: 8w7s
TitleYeast replisome in state IV
Components
  • (DNA polymerase ...) x 2
  • (DNA replication complex GINS protein ...) x 4
  • (DNA replication licensing factor ...) x 5
  • Cell division control protein 45
  • DNA (71-mer)
  • Minichromosome maintenance protein 5
KeywordsREPLICATION / replisome / complex / DNA replication
Function / homology
Function and homology information


establishment of sister chromatid cohesion / DNA-templated DNA replication maintenance of fidelity / Unwinding of DNA / Cul8-RING ubiquitin ligase complex / DNA replication initiation / MCM core complex / Assembly of the pre-replicative complex / Switching of origins to a post-replicative state / epsilon DNA polymerase complex / DNA strand elongation involved in mitotic DNA replication ...establishment of sister chromatid cohesion / DNA-templated DNA replication maintenance of fidelity / Unwinding of DNA / Cul8-RING ubiquitin ligase complex / DNA replication initiation / MCM core complex / Assembly of the pre-replicative complex / Switching of origins to a post-replicative state / epsilon DNA polymerase complex / DNA strand elongation involved in mitotic DNA replication / MCM complex binding / GINS complex / nuclear DNA replication / mitotic DNA replication preinitiation complex assembly / premeiotic DNA replication / pre-replicative complex assembly involved in nuclear cell cycle DNA replication / mitotic DNA replication / Activation of the pre-replicative complex / CMG complex / nuclear pre-replicative complex / Activation of ATR in response to replication stress / DNA replication preinitiation complex / MCM complex / replication fork protection complex / Termination of translesion DNA synthesis / double-strand break repair via break-induced replication / mitotic DNA replication initiation / single-stranded DNA helicase activity / regulation of DNA-templated DNA replication initiation / DNA strand elongation involved in DNA replication / silent mating-type cassette heterochromatin formation / mitotic sister chromatid cohesion / 3'-5' DNA helicase activity / nuclear chromosome / DNA unwinding involved in DNA replication / nuclear replication fork / DNA replication origin binding / Dual incision in TC-NER / DNA replication initiation / subtelomeric heterochromatin formation / error-prone translesion synthesis / DNA helicase activity / helicase activity / transcription elongation by RNA polymerase II / heterochromatin formation / DNA-templated DNA replication / nucleosome assembly / mitotic cell cycle / single-stranded DNA binding / DNA helicase / chromosome, telomeric region / hydrolase activity / DNA repair / chromatin binding / ATP hydrolysis activity / DNA binding / nucleoplasm / ATP binding / identical protein binding / nucleus / metal ion binding / cytoplasm
Similarity search - Function
DNA polymerase epsilon, subunit B / : / DNA polymerase alpha-binding protein Ctf4, C-terminal domain / Minichromosome loss protein Mcl1, middle region / Minichromosome loss protein, Mcl1, middle region / : / MCM3 winged helix domain / GINS/PriA/YqbF domain / CDC45 family / DNA replication complex GINS protein Psf2 ...DNA polymerase epsilon, subunit B / : / DNA polymerase alpha-binding protein Ctf4, C-terminal domain / Minichromosome loss protein Mcl1, middle region / Minichromosome loss protein, Mcl1, middle region / : / MCM3 winged helix domain / GINS/PriA/YqbF domain / CDC45 family / DNA replication complex GINS protein Psf2 / CDC45-like protein / GINS complex, subunit Psf1 / GINS complex, subunit Psf3 / GINS complex, subunit Psf3 superfamily / DNA replication complex GINS protein SLD5, C-terminal / GINS, helical bundle-like domain superfamily / GINS complex protein Sld5, alpha-helical domain / DNA replication complex GINS protein SLD5 C-terminus / GINS complex subunit Sld5 / GINS subunit, domain A / GINS complex protein helical bundle domain / MCM4, winged helix domain / DNA polymerase alpha/delta/epsilon, subunit B / DNA polymerase alpha/epsilon subunit B / : / MCM5, C-terminal domain / DNA replication licensing factor Mcm5 / DNA replication licensing factor Mcm3 / Mini-chromosome maintenance complex protein 4 / DNA replication licensing factor Mcm6 / DNA replication licensing factor Mcm7 / Mcm6, C-terminal winged-helix domain / MCM6 C-terminal winged-helix domain / DNA replication licensing factor Mcm2 / Mini-chromosome maintenance protein 2 / Mini-chromosome maintenance, conserved site / MCM family signature. / MCM N-terminal domain / MCM N-terminal domain / MCM OB domain / Mini-chromosome maintenance protein / MCM, AAA-lid domain / MCM P-loop domain / MCM OB domain / MCM AAA-lid domain / MCM family domain profile. / minichromosome maintenance proteins / MCM domain / WD40-repeat-containing domain superfamily / Winged helix-like DNA-binding domain superfamily / WD40/YVTN repeat-like-containing domain superfamily / ATPases associated with a variety of cellular activities / AAA+ ATPase domain / Nucleic acid-binding, OB-fold / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
DNA / DNA (> 10) / DNA replication licensing factor MCM2 / DNA replication licensing factor MCM3 / DNA polymerase epsilon subunit B / Minichromosome maintenance protein 5 / DNA replication licensing factor MCM4 / DNA replication licensing factor MCM7 / DNA replication complex GINS protein PSF2 / DNA replication licensing factor MCM6 ...DNA / DNA (> 10) / DNA replication licensing factor MCM2 / DNA replication licensing factor MCM3 / DNA polymerase epsilon subunit B / Minichromosome maintenance protein 5 / DNA replication licensing factor MCM4 / DNA replication licensing factor MCM7 / DNA replication complex GINS protein PSF2 / DNA replication licensing factor MCM6 / DNA polymerase alpha-binding protein / DNA replication complex GINS protein SLD5 / Cell division control protein 45 / DNA replication complex GINS protein PSF3 / DNA replication complex GINS protein PSF1
Similarity search - Component
Biological speciesSaccharomyces cerevisiae (brewer's yeast)
Saccharomyces cerevisiae S288C (yeast)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 7.39 Å
AuthorsDang, S. / Zhai, Y. / Feng, J. / Yu, D.
Funding support Hong Kong, 8items
OrganizationGrant numberCountry
The University Grants Committee, Research Grants Council (RGC)GRF16104617 Hong Kong
The University Grants Committee, Research Grants Council (RGC)GRF16103918 Hong Kong
The University Grants Committee, Research Grants Council (RGC)GRF17112119 Hong Kong
The University Grants Committee, Research Grants Council (RGC)GRF17101720 Hong Kong
The University Grants Committee, Research Grants Council (RGC)C7028-19GF Hong Kong
The University Grants Committee, Research Grants Council (RGC)C7009-20GF Hong Kong
The University Grants Committee, Research Grants Council (RGC)ECS26101919 Hong Kong
The University Grants Committee, Research Grants Council (RGC)GRF16103321 Hong Kong
CitationJournal: Nat Commun / Year: 2023
Title: Synergism between CMG helicase and leading strand DNA polymerase at replication fork.
Authors: Zhichun Xu / Jianrong Feng / Daqi Yu / Yunjing Huo / Xiaohui Ma / Wai Hei Lam / Zheng Liu / Xiang David Li / Toyotaka Ishibashi / Shangyu Dang / Yuanliang Zhai /
Abstract: The replisome that replicates the eukaryotic genome consists of at least three engines: the Cdc45-MCM-GINS (CMG) helicase that separates duplex DNA at the replication fork and two DNA polymerases, ...The replisome that replicates the eukaryotic genome consists of at least three engines: the Cdc45-MCM-GINS (CMG) helicase that separates duplex DNA at the replication fork and two DNA polymerases, one on each strand, that replicate the unwound DNA. Here, we determined a series of cryo-electron microscopy structures of a yeast replisome comprising CMG, leading-strand polymerase Polε and three accessory factors on a forked DNA. In these structures, Polε engages or disengages with the motor domains of the CMG by occupying two alternative positions, which closely correlate with the rotational movement of the single-stranded DNA around the MCM pore. During this process, the polymerase remains stably coupled to the helicase using Psf1 as a hinge. This synergism is modulated by a concerted rearrangement of ATPase sites to drive DNA translocation. The Polε-MCM coupling is not only required for CMG formation to initiate DNA replication but also facilitates the leading-strand DNA synthesis mediated by Polε. Our study elucidates a mechanism intrinsic to the replisome that coordinates the activities of CMG and Polε to negotiate any roadblocks, DNA damage, and epigenetic marks encountered during translocation along replication forks.
History
DepositionAug 31, 2023Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Dec 20, 2023Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
2: DNA replication licensing factor MCM2
3: DNA replication licensing factor MCM3
4: DNA replication licensing factor MCM4
5: Minichromosome maintenance protein 5
6: DNA replication licensing factor MCM6
7: DNA replication licensing factor MCM7
A: DNA replication complex GINS protein PSF1
B: DNA replication complex GINS protein PSF2
C: DNA replication complex GINS protein PSF3
D: DNA replication complex GINS protein SLD5
E: Cell division control protein 45
F: DNA polymerase alpha-binding protein
G: DNA polymerase alpha-binding protein
H: DNA polymerase alpha-binding protein
I: DNA (71-mer)
N: DNA polymerase epsilon subunit B


Theoretical massNumber of molelcules
Total (without water)1,200,17616
Polymers1,200,17616
Non-polymers00
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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DNA replication licensing factor ... , 5 types, 5 molecules 23467

#1: Protein DNA replication licensing factor MCM2


Mass: 98911.539 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (brewer's yeast)
Gene: MCM2 / Production host: Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: A0A6A5Q1S9
#2: Protein DNA replication licensing factor MCM3 / Minichromosome maintenance protein 3


Mass: 107653.508 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae S288C (yeast) / Strain: ATCC 204508 / S288c / Gene: MCM3 / Production host: Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: P24279, DNA helicase
#3: Protein DNA replication licensing factor MCM4 / Cell division control protein 54


Mass: 105138.375 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae S288C (yeast) / Strain: ATCC 204508 / S288c / Gene: MCM4 / Production host: Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: P30665, DNA helicase
#5: Protein DNA replication licensing factor MCM6 / Minichromosome maintenance protein 6


Mass: 113110.211 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae S288C (yeast) / Strain: ATCC 204508 / S288c / Gene: MCM6 / Production host: Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: P53091, DNA helicase
#6: Protein DNA replication licensing factor MCM7 / Cell division control protein 47 / Minichromosome maintenance protein 7


Mass: 95049.875 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae S288C (yeast) / Strain: ATCC 204508 / S288c / Gene: MCM7 / Production host: Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: P38132, DNA helicase

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Protein , 2 types, 2 molecules 5E

#4: Protein Minichromosome maintenance protein 5 / Cell division control protein 46


Mass: 86505.734 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae S288C (yeast) / Strain: ATCC 204508 / S288c / Gene: MCM5 / Production host: Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: P29496, DNA helicase
#11: Protein Cell division control protein 45


Mass: 74324.836 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae S288C (yeast) / Strain: ATCC 204508 / S288c / Gene: CDC45 / Production host: Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: Q08032

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DNA replication complex GINS protein ... , 4 types, 4 molecules ABCD

#7: Protein DNA replication complex GINS protein PSF1


Mass: 24230.576 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae S288C (yeast) / Strain: ATCC 204508 / S288c / Gene: PSF1 / Production host: Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: Q12488
#8: Protein DNA replication complex GINS protein PSF2


Mass: 25096.807 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae S288C (yeast) / Strain: ATCC 204508 / S288c / Gene: PSF2 / Production host: Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: P40359
#9: Protein DNA replication complex GINS protein PSF3


Mass: 21977.135 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae S288C (yeast) / Strain: ATCC 204508 / S288c / Gene: PSF3 / Production host: Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: Q12146
#10: Protein DNA replication complex GINS protein SLD5


Mass: 33983.617 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae S288C (yeast) / Strain: ATCC 204508 / S288c / Gene: SLD5 / Production host: Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: Q03406

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DNA polymerase ... , 2 types, 4 molecules FGHN

#12: Protein DNA polymerase alpha-binding protein


Mass: 104543.391 Da / Num. of mol.: 3
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae S288C (yeast) / Strain: ATCC 204508 / S288c / Gene: CTF4 / Production host: Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: Q01454
#14: Protein DNA polymerase epsilon subunit B


Mass: 78425.852 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae S288C (yeast) / Strain: ATCC 204508 / S288c / Gene: DPB2 / Production host: Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: P24482

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DNA chain , 1 types, 1 molecules I

#13: DNA chain DNA (71-mer)


Mass: 22138.150 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Saccharomyces cerevisiae (brewer's yeast)

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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: replisome complex in state IV / Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Molecular weightValue: 1.70 MDa / Experimental 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.NameFormulaBuffer-ID
1100 mMsodium potassiumKCl1
225 mM4-(2-hydroxyethyl)-1-piperazineethanesulfonic acidHEPES1
34 mMmagnesium chlorideMgCl21
40.5 mMAdenosine 5'-O-(3-thio)triphosphateATP-gamma-S1
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: C-flat-1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277.15 K
Details: blot with filter paper for 3-4 seconds before plunging.

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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 magnification: 81000 X / Nominal defocus max: 2500 nm / Nominal defocus min: 1000 nm / Cs: 2.7 mm / C2 aperture diameter: 100 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 4.5 sec. / Electron dose: 53 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Num. of grids imaged: 4 / Num. of real images: 21776
Details: Images were collected in movie-mode containing 40 frames.
EM imaging opticsEnergyfilter name: GIF Bioquantum / Energyfilter slit width: 20 eV
Image scansSampling size: 5 µm / Width: 5760 / Height: 4092

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Processing

EM software
IDNameVersionCategory
1Gautomatchv0.56particle selection
2EPU2.10.0image acquisition
4Gctfv1.18CTF correction
7UCSF Chimeramodel fitting
9PHENIXmodel refinement
10Cootmodel refinement
11cryoSPARCv3.0.1initial Euler assignment
12cryoSPARCv2.15.0initial Euler assignment
13cryoSPARCv3.0.1final Euler assignment
14cryoSPARCv2.15.0final Euler assignment
16cryoSPARCv3.0.13D reconstruction
17cryoSPARCv2.15.03D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 7.39 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 7939 / Algorithm: EXACT BACK PROJECTION / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingProtocol: AB INITIO MODEL / Space: REAL
Atomic model buildingPDB-ID: 6SKL

6skl


Accession code: 6SKL / Source name: PDB / Type: experimental model
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.01151883
ELECTRON MICROSCOPYf_angle_d1.24970232
ELECTRON MICROSCOPYf_dihedral_angle_d16.39619503
ELECTRON MICROSCOPYf_chiral_restr0.1198034
ELECTRON MICROSCOPYf_plane_restr0.0068949

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