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- PDB-8q6p: X. laevis CMG dimer bound to dimeric DONSON - MCM ATPase -

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Basic information

Entry
Database: PDB / ID: 8q6p
TitleX. laevis CMG dimer bound to dimeric DONSON - MCM ATPase
Components
  • (DNA replication licensing factor ...) x 4
  • (Maternal DNA replication licensing factor ...) x 2
  • Protein downstream neighbor of son homolog
KeywordsREPLICATION / DNA replication initiation / Xenopus egg extract / primordial dwarfism / replicative helicase / genome stability
Function / homology
Function and homology information


premeiotic DNA replication / CMG complex / MCM complex / mitotic DNA replication checkpoint signaling / replisome / regulation of DNA-templated DNA replication initiation / replication fork processing / DNA unwinding involved in DNA replication / DNA replication origin binding / mitotic G2 DNA damage checkpoint signaling ...premeiotic DNA replication / CMG complex / MCM complex / mitotic DNA replication checkpoint signaling / replisome / regulation of DNA-templated DNA replication initiation / replication fork processing / DNA unwinding involved in DNA replication / DNA replication origin binding / mitotic G2 DNA damage checkpoint signaling / DNA replication initiation / DNA helicase activity / replication fork / DNA damage checkpoint signaling / helicase activity / DNA helicase / DNA replication / cell cycle / cell division / chromatin binding / chromatin / ATP hydrolysis activity / DNA binding / ATP binding / metal ion binding / nucleus
Similarity search - Function
Donson / 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 ...Donson / 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 / 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
ADENOSINE-5'-TRIPHOSPHATE / DNA replication licensing factor mcm4-B / Maternal DNA replication licensing factor mcm3 / DNA replication licensing factor mcm2 / DNA replication licensing factor mcm5-A / Maternal DNA replication licensing factor mcm6 / Protein downstream neighbor of son homolog / DNA replication licensing factor mcm7-B
Similarity search - Component
Biological speciesXenopus laevis (African clawed frog)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.53 Å
AuthorsButryn, A. / Cvetkovic, M.A. / Costa, A.
Funding support United Kingdom, European Union, 5items
OrganizationGrant numberCountry
The Francis Crick InstituteFC001065 United Kingdom
European Research Council (ERC)820102European Union
Marie Sklodowska-Curie Actions, FragNET ITN845939European Union
Wellcome Trust215510/Z/19/Z United Kingdom
Biotechnology and Biological Sciences Research Council (BBSRC)BB/T001860/1 United Kingdom
CitationJournal: Mol Cell / Year: 2023
Title: The structural mechanism of dimeric DONSON in replicative helicase activation.
Authors: Milos A Cvetkovic / Paolo Passaretti / Agata Butryn / Alicja Reynolds-Winczura / Georgia Kingsley / Aggeliki Skagia / Cyntia Fernandez-Cuesta / Divyasree Poovathumkadavil / Roger George / ...Authors: Milos A Cvetkovic / Paolo Passaretti / Agata Butryn / Alicja Reynolds-Winczura / Georgia Kingsley / Aggeliki Skagia / Cyntia Fernandez-Cuesta / Divyasree Poovathumkadavil / Roger George / Anoop S Chauhan / Satpal S Jhujh / Grant S Stewart / Agnieszka Gambus / Alessandro Costa /
Abstract: The MCM motor of the replicative helicase is loaded onto origin DNA as an inactive double hexamer before replication initiation. Recruitment of activators GINS and Cdc45 upon S-phase transition ...The MCM motor of the replicative helicase is loaded onto origin DNA as an inactive double hexamer before replication initiation. Recruitment of activators GINS and Cdc45 upon S-phase transition promotes the assembly of two active CMG helicases. Although work with yeast established the mechanism for origin activation, how CMG is formed in higher eukaryotes is poorly understood. Metazoan Downstream neighbor of Son (DONSON) has recently been shown to deliver GINS to MCM during CMG assembly. What impact this has on the MCM double hexamer is unknown. Here, we used cryoelectron microscopy (cryo-EM) on proteins isolated from replicating Xenopus egg extracts to identify a double CMG complex bridged by a DONSON dimer. We find that tethering elements mediating complex formation are essential for replication. DONSON reconfigures the MCM motors in the double CMG, and primordial dwarfism patients' mutations disrupting DONSON dimerization affect GINS and MCM engagement in human cells and DNA synthesis in Xenopus egg extracts.
History
DepositionAug 14, 2023Deposition site: PDBE / Processing site: PDBE
Revision 1.0Oct 18, 2023Provider: repository / Type: Initial release
Revision 1.1Oct 25, 2023Group: Database references / Category: citation / Item: _citation.pdbx_database_id_PubMed / _citation.title
Revision 1.2Nov 29, 2023Group: Database references / Category: citation / Item: _citation.journal_volume / _citation.page_first

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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: Maternal DNA replication licensing factor mcm3
4: DNA replication licensing factor mcm4-B
5: DNA replication licensing factor mcm5-A
6: Maternal DNA replication licensing factor mcm6
7: DNA replication licensing factor mcm7-B
L: Protein downstream neighbor of son homolog
hetero molecules


Theoretical massNumber of molelcules
Total (without water)611,74212
Polymers609,2067
Non-polymers2,5365
Water0
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 ... , 4 types, 4 molecules 2457

#1: Protein DNA replication licensing factor mcm2 / BM28-homolog / Minichromosome maintenance protein 2 / xMCM2 / p112


Mass: 100397.648 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Xenopus laevis (African clawed frog) / References: UniProt: P55861, DNA helicase
#3: Protein DNA replication licensing factor mcm4-B / CDC21 homolog-B / Minichromosome maintenance protein 4-B / xMCM4-B / P1-CDC21-B


Mass: 97256.305 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Xenopus laevis (African clawed frog) / References: UniProt: P30664, DNA helicase
#4: Protein DNA replication licensing factor mcm5-A / xMCM5-A / CDC46 homolog A / xCDC46-A / CDC46p / p92


Mass: 82556.414 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Xenopus laevis (African clawed frog) / References: UniProt: P55862, DNA helicase
#6: Protein DNA replication licensing factor mcm7-B / CDC47 homolog B / CDC47-2p / Minichromosome maintenance protein 7-B / xMCM7-B


Mass: 81841.539 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Xenopus laevis (African clawed frog) / References: UniProt: Q7ZXB1, DNA helicase

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Maternal DNA replication licensing factor ... , 2 types, 2 molecules 36

#2: Protein Maternal DNA replication licensing factor mcm3 / Maternal minichromosome maintenance protein 3 / mMCM3 / xMCM3 / P1 homolog / XRLF subunit beta / p100


Mass: 90536.500 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Xenopus laevis (African clawed frog) / References: UniProt: P49739, DNA helicase
#5: Protein Maternal DNA replication licensing factor mcm6 / Maternal minichromosome maintenance protein 6 / mMCM6 / xMCM6


Mass: 92752.969 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Xenopus laevis (African clawed frog) / References: UniProt: Q5FWY4, DNA helicase

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Protein / Non-polymers , 2 types, 6 molecules L

#7: Protein Protein downstream neighbor of son homolog


Mass: 63864.559 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Xenopus laevis (African clawed frog) / References: UniProt: Q5U4U4
#8: Chemical
ChemComp-ATP / ADENOSINE-5'-TRIPHOSPHATE / Adenosine triphosphate


Mass: 507.181 Da / Num. of mol.: 5 / Source method: obtained synthetically / Formula: C10H16N5O13P3 / Comment: ATP, energy-carrying molecule*YM

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Details

Has ligand of interestN

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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: X. laevis CMG dimer bound to dimeric DONSON / Type: COMPLEX / Entity ID: #1-#7 / Source: NATURAL
Molecular weightValue: 1.54 MDa / Experimental value: NO
Source (natural)Organism: Xenopus laevis (African clawed frog)
Buffer solutionpH: 7.5
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE

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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 FIELDBright-field microscopy / Nominal defocus max: 3400 nm / Nominal defocus min: 1300 nm
Image recordingElectron dose: 33 e/Å2 / Film or detector model: FEI FALCON IV (4k x 4k)

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Processing

CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.53 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 144989 / Symmetry type: POINT
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 121.67 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.001817031
ELECTRON MICROSCOPYf_angle_d0.396522970
ELECTRON MICROSCOPYf_chiral_restr0.03632613
ELECTRON MICROSCOPYf_plane_restr0.00272931
ELECTRON MICROSCOPYf_dihedral_angle_d5.02632304

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