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- PDB-9e2x: Cryo-EM structure of yeast CMG helicase stalled at G4-containing ... -

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Basic information

Entry
Database: PDB / ID: 9e2x
TitleCryo-EM structure of yeast CMG helicase stalled at G4-containing DNA template, state 2
Components
  • (DNA replication complex GINS protein ...) x 4
  • (DNA replication licensing factor ...) x 5
  • Cell division control protein 45
  • Chromosome segregation in meiosis protein 3
  • Lagging strand DNA template
  • Leading strand DNA template
  • Minichromosome maintenance protein 5
  • Topoisomerase 1-associated factor 1
KeywordsREPLICATION/DNA / helicase / DNA replication / cell division / fork stalling / translocation / REPLICATION / REPLICATION-DNA complex
Function / homology
Function and homology information


Unwinding of DNA / maintenance of DNA repeat elements / replication fork arrest / meiotic chromosome segregation / DNA strand elongation involved in mitotic DNA replication / GINS complex / MCM core complex / Assembly of the pre-replicative complex / Switching of origins to a post-replicative state / MCM complex binding ...Unwinding of DNA / maintenance of DNA repeat elements / replication fork arrest / meiotic chromosome segregation / DNA strand elongation involved in mitotic DNA replication / GINS complex / MCM core complex / Assembly of the pre-replicative complex / Switching of origins to a post-replicative state / MCM complex binding / mitotic DNA replication preinitiation complex assembly / nuclear DNA replication / premeiotic DNA replication / pre-replicative complex assembly involved in nuclear cell cycle DNA replication / mitotic DNA replication / Activation of the pre-replicative complex / DNA replication checkpoint signaling / CMG complex / establishment of mitotic sister chromatid cohesion / nuclear pre-replicative complex / Activation of ATR in response to replication stress / DNA replication preinitiation complex / MCM complex / replication fork protection complex / double-strand break repair via break-induced replication / single-stranded DNA helicase activity / mitotic DNA replication initiation / regulation of DNA-templated DNA replication initiation / silent mating-type cassette heterochromatin formation / mitotic sister chromatid cohesion / DNA strand elongation involved in DNA replication / replication fork processing / nuclear replication fork / DNA replication origin binding / DNA replication initiation / subtelomeric heterochromatin formation / meiotic cell cycle / helicase activity / transcription elongation by RNA polymerase II / DNA-templated DNA replication / heterochromatin formation / single-stranded DNA binding / DNA helicase / forked DNA-dependent helicase activity / single-stranded 3'-5' DNA helicase activity / four-way junction helicase activity / double-stranded DNA helicase activity / chromosome, telomeric region / DNA replication / DNA repair / DNA damage response / chromatin binding / ATP hydrolysis activity / DNA binding / zinc ion binding / nucleoplasm / ATP binding / nucleus / cytosol / cytoplasm
Similarity search - Function
Chromosome segregation in meiosis protein 3 / TIPIN/Csm3/Swi3 / Replication Fork Protection Component Swi3 / Timeless, N-terminal / Timeless / Timeless protein / : / PSF3 N-terminal domain / CDC45 family / DNA replication complex GINS protein Psf2 ...Chromosome segregation in meiosis protein 3 / TIPIN/Csm3/Swi3 / Replication Fork Protection Component Swi3 / Timeless, N-terminal / Timeless / Timeless protein / : / PSF3 N-terminal domain / CDC45 family / DNA replication complex GINS protein Psf2 / CDC45 / GINS complex, subunit Psf1 / GINS complex, subunit Psf3 / DNA replication complex GINS protein SLD5, C-terminal / GINS, helical bundle-like domain superfamily / GINS complex, subunit Psf3 superfamily / GINS complex protein Sld5, alpha-helical domain / : / DNA replication complex GINS protein SLD5 C-terminus / MCM3 winged helix domain / GINS complex subunit Sld5 / GINS subunit, domain A / GINS complex protein helical bundle domain / MCM4, winged helix domain / : / MCM5, C-terminal domain / DNA replication licensing factor Mcm5 / MCM3-like, winged helix domain / 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 / MCM OB domain / Mini-chromosome maintenance protein / MCM, AAA-lid domain / MCM P-loop domain / MCM AAA-lid domain / MCM family domain profile. / minichromosome maintenance proteins / MCM domain / Winged helix-like DNA-binding domain superfamily / Nucleic acid-binding, OB-fold / ATPases associated with a variety of cellular activities / AAA+ ATPase domain / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
ADENOSINE-5'-DIPHOSPHATE / ADENOSINE-5'-TRIPHOSPHATE / DNA / DNA (> 10) / DNA replication licensing factor MCM3 / DNA replication licensing factor MCM2 / Minichromosome maintenance protein 5 / DNA replication licensing factor MCM4 / DNA replication licensing factor MCM7 / DNA replication complex GINS protein PSF2 ...ADENOSINE-5'-DIPHOSPHATE / ADENOSINE-5'-TRIPHOSPHATE / DNA / DNA (> 10) / DNA replication licensing factor MCM3 / DNA replication licensing factor MCM2 / 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 / Topoisomerase 1-associated factor 1 / DNA replication complex GINS protein SLD5 / Chromosome segregation in meiosis protein 3 / Cell division control protein 45 / DNA replication complex GINS protein PSF3 / DNA replication complex GINS protein PSF1
Similarity search - Component
Biological speciesSaccharomyces cerevisiae W303 (yeast)
synthetic construct (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.5 Å
AuthorsAllwein, B. / Batra, S. / Remus, D. / Hite, R.
Funding support United States, 3items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R35GM152094 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R35GM126907 United States
National Institutes of Health/National Cancer Institute (NIH/NCI)P30 CA008748 United States
CitationJournal: Science / Year: 2025
Title: G-quadruplex-stalled eukaryotic replisome structure reveals helical inchworm DNA translocation.
Authors: Sahil Batra / Benjamin Allwein / Charanya Kumar / Sujan Devbhandari / Jan-Gert Brüning / Soon Bahng / Chong M Lee / Kenneth J Marians / Richard K Hite / Dirk Remus /
Abstract: DNA G-quadruplexes (G4s) are non-B-form DNA secondary structures that threaten genome stability by impeding DNA replication. To elucidate how G4s induce replication fork arrest, we characterized fork ...DNA G-quadruplexes (G4s) are non-B-form DNA secondary structures that threaten genome stability by impeding DNA replication. To elucidate how G4s induce replication fork arrest, we characterized fork collisions with preformed G4s in the parental DNA using reconstituted yeast and human replisomes. We demonstrate that a single G4 in the leading strand template is sufficient to stall replisomes by arresting the CMG helicase. Cryo-electron microscopy structures of stalled yeast and human CMG complexes reveal that the folded G4 is lodged inside the central CMG channel, arresting translocation. The G4 stabilizes the CMG at distinct translocation intermediates, suggesting an unprecedented helical inchworm mechanism for DNA translocation. These findings illuminate the eukaryotic replication fork mechanism under normal and perturbed conditions.
History
DepositionOct 23, 2024Deposition site: RCSB / Processing site: RCSB
Revision 1.0Mar 19, 2025Provider: repository / Type: Initial release
Revision 1.0Mar 19, 2025Data content type: EM metadata / Data content type: EM metadata / Provider: repository / Type: Initial release
Revision 1.0Mar 19, 2025Data content type: FSC / Data content type: FSC / Provider: repository / Type: Initial release
Revision 1.0Mar 19, 2025Data content type: Image / Data content type: Image / Provider: repository / Type: Initial release
Revision 1.0Mar 19, 2025Data content type: Mask / Part number: 1 / Data content type: Mask / Provider: repository / Type: Initial release
Revision 1.0Mar 19, 2025Data content type: Primary map / Data content type: Primary map / Provider: repository / Type: Initial release
Revision 1.1Mar 26, 2025Group: Data collection / Database references / Category: citation / citation_author / em_admin
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_ASTM / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year / _em_admin.last_update

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
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: Leading strand DNA template
G: Lagging strand DNA template
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
X: Topoisomerase 1-associated factor 1
Y: Chromosome segregation in meiosis protein 3
hetero molecules


Theoretical massNumber of molelcules
Total (without water)965,18431
Polymers961,85215
Non-polymers3,33216
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

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

#1: Protein DNA replication complex GINS protein PSF1 / Partner of Sld five 1


Mass: 24230.576 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae W303 (yeast) / Gene: PSF1, YDR013W, PZA208, YD8119.18 / Production host: Saccharomyces cerevisiae W303 (yeast) / References: UniProt: Q12488
#2: Protein DNA replication complex GINS protein PSF2 / Partner of Sld five 2


Mass: 25096.807 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae W303 (yeast) / Gene: PSF2, YJL072C, HRF213, J1086 / Production host: Saccharomyces cerevisiae W303 (yeast) / References: UniProt: P40359
#3: Protein DNA replication complex GINS protein PSF3 / Partner of Sld five 3


Mass: 24437.859 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae W303 (yeast) / Gene: PSF3, YOL146W / Production host: Saccharomyces cerevisiae W303 (yeast) / References: UniProt: Q12146
#4: 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 W303 (yeast) / Gene: SLD5, YDR489W / Production host: Saccharomyces cerevisiae W303 (yeast) / References: UniProt: Q03406

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Protein , 4 types, 4 molecules E5XY

#5: 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 W303 (yeast) / Gene: CDC45, SLD4, YLR103C, L8004.11 / Production host: Saccharomyces cerevisiae W303 (yeast) / References: UniProt: Q08032
#11: 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 W303 (yeast) / Gene: MCM5, CDC46, YLR274W, L9328.1 / Production host: Saccharomyces cerevisiae W303 (yeast) / References: UniProt: P29496, DNA helicase
#14: Protein Topoisomerase 1-associated factor 1


Mass: 141296.875 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae W303 (yeast) / Gene: TOF1, YNL273W, N0636 / Production host: Saccharomyces cerevisiae W303 (yeast) / References: UniProt: P53840
#15: Protein Chromosome segregation in meiosis protein 3


Mass: 10939.873 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae W303 (yeast) / Gene: CSM3, YMR048W, YM9796.01 / Production host: Saccharomyces cerevisiae W303 (yeast) / References: UniProt: Q04659

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DNA chain , 2 types, 2 molecules FG

#6: DNA chain Leading strand DNA template


Mass: 15060.594 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) synthetic construct (others) / Production host: synthetic construct (others)
#7: DNA chain Lagging strand DNA template


Mass: 6111.983 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) synthetic construct (others) / Production host: synthetic construct (others)

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DNA replication licensing factor ... , 5 types, 5 molecules 23467

#8: Protein DNA replication licensing factor MCM2 / Minichromosome maintenance protein 2


Mass: 98911.539 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae W303 (yeast) / Gene: MCM2, YBL023C, YBL0438 / Production host: Saccharomyces cerevisiae W303 (yeast) / References: UniProt: P29469, DNA helicase
#9: 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 W303 (yeast) / Gene: MCM3, YEL032W, SYGP-ORF23 / Production host: Saccharomyces cerevisiae W303 (yeast) / References: UniProt: P24279, DNA helicase
#10: 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 W303 (yeast) / Gene: MCM4, CDC54, HCD21, YPR019W, YP9531.13 / Production host: Saccharomyces cerevisiae W303 (yeast) / References: UniProt: P30665, DNA helicase
#12: 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 W303 (yeast) / Gene: MCM6, YGL201C / Production host: Saccharomyces cerevisiae W303 (yeast) / References: UniProt: P53091, DNA helicase
#13: 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 W303 (yeast) / Gene: MCM7, CDC47, YBR202W, YBR1441 / Production host: Saccharomyces cerevisiae W303 (yeast) / References: UniProt: P38132, DNA helicase

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Non-polymers , 4 types, 16 molecules

#16: Chemical
ChemComp-ATP / ADENOSINE-5'-TRIPHOSPHATE


Mass: 507.181 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: C10H16N5O13P3 / Feature type: SUBJECT OF INVESTIGATION / Comment: ATP, energy-carrying molecule*YM
#17: Chemical
ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 5 / Source method: obtained synthetically / Formula: Mg
#18: Chemical
ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 5 / Source method: obtained synthetically / Formula: Zn
#19: Chemical ChemComp-ADP / ADENOSINE-5'-DIPHOSPHATE


Mass: 427.201 Da / Num. of mol.: 2 / 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
Has protein modificationY

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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: Yeast CMG-CTM G4 stall state 2 / Type: COMPLEX
Details: Yeast CMG stalled at G4-containing DNA substrate, in a different conformation from state 1.
Entity ID: #8-#13, #1-#7, #14-#15 / Source: RECOMBINANT
Molecular weightValue: 0.70226 MDa / Experimental value: NO
Source (natural)Organism: Saccharomyces cerevisiae W303 (yeast)
Source (recombinant)Organism: Saccharomyces cerevisiae W303 (yeast)
Buffer solutionpH: 7.6
Buffer component
IDConc.NameFormulaBuffer-ID
120 mMHEPES-KOHC8H17KN2O4S1
2100 mMPotassium acetateCH3CO2K1
310 mMMagnesium acetateMg(CH3COO)21
42.5 mMDTTC4H10O2S21
5.02 %NP-40SH(C2H4O)nO(C6H4)C9H191
6100 uMAMP-PNPC10H17N6O12P31
75 mMATPC10H16N5O13P31
SpecimenConc.: 0.01 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid mesh size: 400 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 95 % / Chamber temperature: 277 K
Details: Wait time 30 seconds after sample application; blot time 30 seconds with blot force 0

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: TFS KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 29000 X / Nominal defocus max: 1800 nm / Nominal defocus min: 800 nm / Cs: 2.7 mm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 3 sec. / Electron dose: 66 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 3975

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Processing

EM software
IDNameVersionCategory
1cryoSPARC4.0.0particle selection
2SerialEMimage acquisition
4cryoSPARC4.0.0CTF correction
7Coot0.9.8.91model fitting
8UCSF ChimeraX1.7model fitting
10cryoSPARC4.0.0initial Euler assignment
11cryoSPARC4.0.0final Euler assignment
12cryoSPARC4.0.0classification
13cryoSPARC4.0.03D reconstruction
14PHENIX1.21-5207model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 1895342
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.5 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 40714 / Symmetry type: POINT
Atomic model buildingB value: 82.32 / Protocol: AB INITIO MODEL / Space: REAL
Details: Initial fitting was performed de novo by ModelAngelo, then iteratively improved with ChimeraX/ISOLDE, Coot, and Phenix real-space refinement algorithms.
Atomic model buildingDetails: ModelAngelo / Source name: Other / Type: in silico model

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