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- PDB-9e2z: Cryo-EM structure of human CMG helicase stalled at G4-containing ... -

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Basic information

Entry
Database: PDB / ID: 9e2z
TitleCryo-EM structure of human CMG helicase stalled at G4-containing DNA template
Components
  • (DNA replication complex GINS protein ...) x 4
  • (DNA replication licensing factor ...) x 5
  • Cell division control protein 45 homolog
  • Isoform 2 of DNA replication licensing factor MCM3
  • Lagging strand DNA template
  • Leading strand DNA template
KeywordsREPLICATION/DNA / helicase / DNA replication / cell division / fork stalling / translocation / REPLICATION / REPLICATION-DNA complex
Function / homology
Function and homology information


Switching of origins to a post-replicative state / Unwinding of DNA / DNA strand elongation involved in mitotic DNA replication / GINS complex / mitotic DNA replication preinitiation complex assembly / nuclear origin of replication recognition complex / alpha DNA polymerase:primase complex / mitotic DNA replication / DNA replication checkpoint signaling / CMG complex ...Switching of origins to a post-replicative state / Unwinding of DNA / DNA strand elongation involved in mitotic DNA replication / GINS complex / mitotic DNA replication preinitiation complex assembly / nuclear origin of replication recognition complex / alpha DNA polymerase:primase complex / mitotic DNA replication / DNA replication checkpoint signaling / CMG complex / regulation of phosphorylation / DNA replication preinitiation complex / MCM complex / double-strand break repair via break-induced replication / mitotic DNA replication initiation / regulation of DNA-templated DNA replication initiation / inner cell mass cell proliferation / DNA strand elongation involved in DNA replication / cochlea development / G1/S-Specific Transcription / : / DNA replication origin binding / Activation of the pre-replicative complex / DNA replication initiation / Activation of ATR in response to replication stress / cellular response to interleukin-4 / forked DNA-dependent helicase activity / single-stranded 3'-5' DNA helicase activity / four-way junction helicase activity / double-stranded DNA helicase activity / cellular response to epidermal growth factor stimulus / ciliary basal body / Assembly of the pre-replicative complex / Orc1 removal from chromatin / cellular response to xenobiotic stimulus / nucleosome assembly / single-stranded DNA binding / histone binding / chromatin extrusion motor activity / ATP-dependent H2AZ histone chaperone activity / ATP-dependent H3-H4 histone complex chaperone activity / DNA helicase / cohesin loader activity / DNA clamp loader activity / DNA replication / chromosome, telomeric region / cell population proliferation / centrosome / DNA damage response / chromatin binding / apoptotic process / chromatin / perinuclear region of cytoplasm / enzyme binding / ATP hydrolysis activity / DNA binding / nucleoplasm / ATP binding / identical protein binding / nucleus / membrane / metal ion binding / cytosol / cytoplasm
Similarity search - Function
: / PSF3 N-terminal domain / CDC45 family / DNA replication complex GINS protein Psf2 / CDC45 / 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 ...: / PSF3 N-terminal domain / CDC45 family / DNA replication complex GINS protein Psf2 / CDC45 / 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 / : / 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 / 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 / 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) / Cell division control protein 45 homolog / DNA replication licensing factor MCM3 / DNA replication licensing factor MCM4 / DNA replication licensing factor MCM5 / DNA replication licensing factor MCM7 ...ADENOSINE-5'-DIPHOSPHATE / ADENOSINE-5'-TRIPHOSPHATE / : / DNA / DNA (> 10) / Cell division control protein 45 homolog / DNA replication licensing factor MCM3 / DNA replication licensing factor MCM4 / DNA replication licensing factor MCM5 / DNA replication licensing factor MCM7 / DNA replication licensing factor MCM2 / DNA replication licensing factor MCM6 / DNA replication complex GINS protein PSF1 / DNA replication complex GINS protein SLD5 / DNA replication complex GINS protein PSF3 / DNA replication complex GINS protein PSF2
Similarity search - Component
Biological speciesHomo sapiens (human)
synthetic construct (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.6 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 26, 2025Provider: 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: Isoform 2 of DNA replication licensing factor MCM3
4: DNA replication licensing factor MCM4
5: DNA replication licensing factor MCM5
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 homolog
F: Leading strand DNA template
G: Lagging strand DNA template
hetero molecules


Theoretical massNumber of molelcules
Total (without water)738,68031
Polymers735,23213
Non-polymers3,44918
Water70339
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 licensing factor ... , 5 types, 5 molecules 24567

#1: Protein DNA replication licensing factor MCM2 / Minichromosome maintenance protein 2 homolog / Nuclear protein BM28


Mass: 102034.102 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: MCM2, BM28, CCNL1, CDCL1, KIAA0030 / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: P49736, DNA helicase
#3: Protein DNA replication licensing factor MCM4 / CDC21 homolog / P1-CDC21


Mass: 99119.461 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: MCM4, CDC21 / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: P33991, DNA helicase
#4: Protein DNA replication licensing factor MCM5 / CDC46 homolog / P1-CDC46


Mass: 82406.633 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: MCM5, CDC46 / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: P33992, DNA helicase
#5: Protein DNA replication licensing factor MCM6 / p105MCM


Mass: 93010.273 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: MCM6 / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: Q14566, DNA helicase
#6: Protein DNA replication licensing factor MCM7 / CDC47 homolog / P1.1-MCM3


Mass: 81411.875 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: MCM7, CDC47, MCM2 / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: P33993, DNA helicase

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Protein , 2 types, 2 molecules 3E

#2: Protein Isoform 2 of DNA replication licensing factor MCM3 / DNA polymerase alpha holoenzyme-associated protein P1 / P1-MCM3 / RLF subunit beta / p102


Mass: 96043.320 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: MCM3 / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: P25205, DNA helicase
#11: Protein Cell division control protein 45 homolog / PORC-PI-1


Mass: 65651.539 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: CDC45, CDC45L, CDC45L2, UNQ374/PRO710 / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: O75419

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

#7: Protein DNA replication complex GINS protein PSF1 / GINS complex subunit 1


Mass: 23022.469 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: GINS1, KIAA0186, PSF1 / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: Q14691
#8: Protein DNA replication complex GINS protein PSF2 / GINS complex subunit 2


Mass: 25336.793 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: GINS2, PSF2, CGI-122, DC5, HSPC037 / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: Q9Y248
#9: Protein DNA replication complex GINS protein PSF3 / GINS complex subunit 3


Mass: 24562.611 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: GINS3, PSF3 / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: Q9BRX5
#10: Protein DNA replication complex GINS protein SLD5 / GINS complex subunit 4


Mass: 26081.873 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: GINS4, SLD5 / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: Q9BRT9

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DNA chain , 2 types, 2 molecules FG

#12: DNA chain Leading strand DNA template


Mass: 12582.038 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) synthetic construct (others) / Production host: synthetic construct (others)
#13: DNA chain Lagging strand DNA template


Mass: 3968.626 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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Non-polymers , 6 types, 57 molecules

#14: 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
#15: Chemical
ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 6 / Source method: obtained synthetically / Formula: Mg
#16: Chemical
ChemComp-ATP / ADENOSINE-5'-TRIPHOSPHATE


Mass: 507.181 Da / Num. of mol.: 5 / Source method: obtained synthetically / Formula: C10H16N5O13P3 / Feature type: SUBJECT OF INVESTIGATION / Comment: ATP, energy-carrying molecule*YM
#17: Chemical
ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: Zn
#18: Chemical ChemComp-K / POTASSIUM ION


Mass: 39.098 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: K
#19: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 39 / Source method: isolated from a natural source / Formula: H2O

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Details

Has ligand of interestY
Has protein modificationN

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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: Human CMG, G4 stall state / Type: COMPLEX
Details: Human CMG stalled at G4-containing DNA template, lacking the fork protection complex (TIMELESS-TIPIN)
Entity ID: #7-#13, #2, #5, #4, #1, #3, #6 / Source: RECOMBINANT
Molecular weightValue: 0.58601 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: 30 second wait time after sample application; 30 second blot time, 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: 3325

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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: 1587336
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 2.6 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 187345 / Symmetry type: POINT
Atomic model buildingB value: 49.99 / 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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