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- PDB-7p5z: Structure of a DNA-loaded MCM double hexamer engaged with the Dbf... -

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Basic information

Entry
Database: PDB / ID: 7p5z
TitleStructure of a DNA-loaded MCM double hexamer engaged with the Dbf4-dependent kinase
Components
  • (DNA (53-MER)) x 2
  • (DNA replication licensing factor ...) x 5
  • Cell division control protein 7
  • DDK kinase regulatory subunit DBF4
  • Minichromosome maintenance protein 5
KeywordsREPLICATION / Kinase / Mcm2-7 helicase / nucleoprotein complex / DNA replication
Function / homology
Function and homology information


positive regulation of spindle attachment to meiosis I kinetochore / positive regulation of meiotic DNA double-strand break formation involved in reciprocal meiotic recombination / positive regulation of kinetochore assembly / positive regulation of meiotic DNA double-strand break formation / positive regulation of DNA replication initiation / negative regulation of exit from mitosis / Dbf4-dependent protein kinase complex / positive regulation of protein localization to kinetochore / positive regulation of meiosis I / positive regulation of nuclear cell cycle DNA replication ...positive regulation of spindle attachment to meiosis I kinetochore / positive regulation of meiotic DNA double-strand break formation involved in reciprocal meiotic recombination / positive regulation of kinetochore assembly / positive regulation of meiotic DNA double-strand break formation / positive regulation of DNA replication initiation / negative regulation of exit from mitosis / Dbf4-dependent protein kinase complex / positive regulation of protein localization to kinetochore / positive regulation of meiosis I / positive regulation of nuclear cell cycle DNA replication / regulation of cell cycle phase transition / 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 / replication fork protection complex / pre-replicative complex assembly involved in nuclear cell cycle DNA replication / Activation of the pre-replicative complex / mitotic DNA replication / CMG complex / nuclear pre-replicative complex / Activation of ATR in response to replication stress / DNA replication preinitiation complex / MCM complex / protein-containing complex localization / mitotic DNA replication checkpoint signaling / double-strand break repair via break-induced replication / mitotic DNA replication initiation / single-stranded DNA helicase activity / silent mating-type cassette heterochromatin formation / regulation of DNA-templated DNA replication initiation / DNA strand elongation involved in DNA replication / nuclear replication fork / DNA replication origin binding / chromosome, centromeric region / DNA replication initiation / subtelomeric heterochromatin formation / DNA helicase activity / protein serine/threonine kinase activator activity / chromosome segregation / transcription elongation by RNA polymerase II / helicase activity / heterochromatin formation / single-stranded DNA binding / DNA helicase / chromosome, telomeric region / DNA replication / non-specific serine/threonine protein kinase / cell division / protein serine kinase activity / protein serine/threonine kinase activity / DNA damage response / centrosome / chromatin binding / chromatin / signal transduction / ATP hydrolysis activity / zinc ion binding / nucleoplasm / ATP binding / metal ion binding / identical protein binding / nucleus / cytoplasm
Similarity search - Function
Regulatory subunit Dfp1/Him1, central region / : / Dfp1/Him1, central region / BRCT domain / Zinc finger, DBF-type / DBF-type zinc finger superfamily / : / DBF zinc finger / Zinc finger DBF4-type profile. / Zinc finger in DBF-like proteins ...Regulatory subunit Dfp1/Him1, central region / : / Dfp1/Him1, central region / BRCT domain / Zinc finger, DBF-type / DBF-type zinc finger superfamily / : / DBF zinc finger / Zinc finger DBF4-type profile. / Zinc finger in DBF-like proteins / : / MCM3 winged helix domain / : / MCM5, C-terminal domain / DNA replication licensing factor MCM7, winged helix / DNA replication licensing factor Mcm5 / MCM4, winged helix domain / DNA replication licensing factor Mcm3 / Mini-chromosome maintenance complex protein 4 / : / MCM3-like, winged helix domain / 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 / BRCT domain superfamily / Serine/threonine-protein kinase, active site / Serine/Threonine protein kinases active-site signature. / Protein kinase domain / Winged helix-like DNA-binding domain superfamily / Serine/Threonine protein kinases, catalytic domain / Nucleic acid-binding, OB-fold / ATPases associated with a variety of cellular activities / AAA+ ATPase domain / Protein kinase domain profile. / Protein kinase domain / Protein kinase-like domain superfamily / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
ADENOSINE-5'-DIPHOSPHATE / ADENOSINE-5'-TRIPHOSPHATE / DNA / DNA (> 10) / Cell division control protein 7 / DNA replication licensing factor MCM3 / DNA replication licensing factor MCM2 / Minichromosome maintenance protein 5 / DNA replication licensing factor MCM4 / DDK kinase regulatory subunit DBF4 ...ADENOSINE-5'-DIPHOSPHATE / ADENOSINE-5'-TRIPHOSPHATE / DNA / DNA (> 10) / Cell division control protein 7 / DNA replication licensing factor MCM3 / DNA replication licensing factor MCM2 / Minichromosome maintenance protein 5 / DNA replication licensing factor MCM4 / DDK kinase regulatory subunit DBF4 / DNA replication licensing factor MCM7 / DNA replication licensing factor MCM6
Similarity search - Component
Biological speciesSaccharomyces cerevisiae (brewer's yeast)
Saccharomyces cerevisiae S288C (yeast)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.3 Å
AuthorsGreiwe, J.F. / Miller, T.C.R. / Martino, F. / Costa, A.
Funding supportEuropean Union, 4items
OrganizationGrant numberCountry
Wellcome TrustFC001065
Medical Research Council (MRC, United Kingdom)FC001065
Cancer Research UKFC001065
European Research Council (ERC)820102European Union
CitationJournal: Nat Struct Mol Biol / Year: 2022
Title: Structural mechanism for the selective phosphorylation of DNA-loaded MCM double hexamers by the Dbf4-dependent kinase.
Authors: Julia F Greiwe / Thomas C R Miller / Julia Locke / Fabrizio Martino / Steven Howell / Anne Schreiber / Andrea Nans / John F X Diffley / Alessandro Costa /
Abstract: Loading of the eukaryotic replicative helicase onto replication origins involves two MCM hexamers forming a double hexamer (DH) around duplex DNA. During S phase, helicase activation requires MCM ...Loading of the eukaryotic replicative helicase onto replication origins involves two MCM hexamers forming a double hexamer (DH) around duplex DNA. During S phase, helicase activation requires MCM phosphorylation by Dbf4-dependent kinase (DDK), comprising Cdc7 and Dbf4. DDK selectively phosphorylates loaded DHs, but how such fidelity is achieved is unknown. Here, we determine the cryogenic electron microscopy structure of Saccharomyces cerevisiae DDK in the act of phosphorylating a DH. DDK docks onto one MCM ring and phosphorylates the opposed ring. Truncation of the Dbf4 docking domain abrogates DH phosphorylation, yet Cdc7 kinase activity is unaffected. Late origin firing is blocked in response to DNA damage via Dbf4 phosphorylation by the Rad53 checkpoint kinase. DDK phosphorylation by Rad53 impairs DH phosphorylation by blockage of DDK binding to DHs, and also interferes with the Cdc7 active site. Our results explain the structural basis and regulation of the selective phosphorylation of DNA-loaded MCM DHs, which supports bidirectional replication.
History
DepositionJul 15, 2021Deposition site: PDBE / Processing site: PDBE
Revision 1.0Feb 2, 2022Provider: repository / Type: Initial release
Revision 1.1Jul 17, 2024Group: Data collection / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / em_3d_fitting_list / em_admin / pdbx_initial_refinement_model
Item: _em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id ..._em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id / _em_3d_fitting_list.source_name / _em_3d_fitting_list.type / _em_admin.last_update

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Structure visualization

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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 licensing factor MCM2
B: DNA replication licensing factor MCM3
C: DNA replication licensing factor MCM4
D: Minichromosome maintenance protein 5
E: DNA replication licensing factor MCM6
F: DNA replication licensing factor MCM7
1: Cell division control protein 7
G: DDK kinase regulatory subunit DBF4
X: DNA (53-MER)
Y: DNA (53-MER)
hetero molecules


Theoretical massNumber of molelcules
Total (without water)1,401,82347
Polymers1,396,42816
Non-polymers5,39531
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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DNA replication licensing factor ... , 5 types, 10 molecules 2A3B4C6E7F

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


Mass: 98911.539 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Strain: ATCC 204508 / S288c / Gene: MCM2, YBL023C, YBL0438 / Production host: Saccharomyces cerevisiae S288C (yeast) / References: UniProt: P29469, DNA helicase
#2: Protein DNA replication licensing factor MCM3 / Minichromosome maintenance protein 3


Mass: 111720.242 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Strain: ATCC 204508 / S288c / Gene: MCM3, YEL032W, SYGP-ORF23 / Production host: Saccharomyces cerevisiae S288C (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.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Strain: ATCC 204508 / S288c / Gene: MCM4, CDC54, HCD21, YPR019W, YP9531.13 / Production host: Saccharomyces cerevisiae S288C (yeast) / References: UniProt: P30665, DNA helicase
#5: Protein DNA replication licensing factor MCM6 / Minichromosome maintenance protein 6


Mass: 113110.211 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Strain: ATCC 204508 / S288c / Gene: MCM6, YGL201C / Production host: Saccharomyces cerevisiae S288C (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.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Strain: ATCC 204508 / S288c / Gene: MCM7, CDC47, YBR202W, YBR1441 / Production host: Saccharomyces cerevisiae S288C (yeast) / References: UniProt: P38132, DNA helicase

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Protein , 3 types, 4 molecules 5D1G

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


Mass: 86505.734 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Strain: ATCC 204508 / S288c / Gene: MCM5, CDC46, YLR274W, L9328.1 / Production host: Saccharomyces cerevisiae S288C (yeast) / References: UniProt: P29496, DNA helicase
#7: Protein Cell division control protein 7


Mass: 58391.129 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Strain: ATCC 204508 / S288c / Gene: CDC7, OAF2, YDL017W, D2855 / Production host: Saccharomyces cerevisiae S288C (yeast)
References: UniProt: P06243, non-specific serine/threonine protein kinase
#8: Protein DDK kinase regulatory subunit DBF4 / Dumbbell forming protein 4


Mass: 84503.305 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Strain: ATCC 204508 / S288c / Gene: DBF4, DNA52, YDR052C, D4205, YD9609.07C / Production host: Saccharomyces cerevisiae S288C (yeast) / References: UniProt: P32325

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DNA chain , 2 types, 2 molecules XY

#9: DNA chain DNA (53-MER)


Mass: 16326.441 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Saccharomyces cerevisiae S288C (yeast)
#10: DNA chain DNA (53-MER)


Mass: 16335.457 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Saccharomyces cerevisiae S288C (yeast)

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Non-polymers , 4 types, 31 molecules

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


Mass: 507.181 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C10H16N5O13P3 / Comment: ATP, energy-carrying molecule*YM
#12: Chemical
ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 10 / Source method: obtained synthetically / Formula: Mg
#13: Chemical
ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 11 / Source method: obtained synthetically / Formula: Zn
#14: Chemical
ChemComp-ADP / ADENOSINE-5'-DIPHOSPHATE


Mass: 427.201 Da / Num. of mol.: 8 / Source method: obtained synthetically / Formula: C10H15N5O10P2 / Comment: ADP, 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: DNA-loaded MCM double hexamer engaged with the dimeric Dbf4-dependent kinase
Type: COMPLEX / Entity ID: #1-#10 / Source: RECOMBINANT
Source (natural)Organism: Saccharomyces cerevisiae S288C (yeast)
Source (recombinant)Organism: Saccharomyces cerevisiae S288C (yeast)
Buffer solutionpH: 7.6
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Details: The entire MCM loading and phosphorylation reaction was applied to the EM grid.
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 90 % / Chamber temperature: 295 K / Details: blotted for 3 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: 130000 X / Nominal defocus max: 4100 nm / Nominal defocus min: 2000 nm / C2 aperture diameter: 50 µm
Specimen holderSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 9 sec. / Electron dose: 51.3 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 18135
EM imaging opticsEnergyfilter name: GIF Bioquantum / Energyfilter slit width: 20 eV
Image scansMovie frames/image: 30

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Processing

Software
NameVersionClassificationNB
phenix.real_space_refine1.19.2_4158refinement
PHENIX1.19.2_4158refinement
EM software
IDNameVersionCategory
1crYOLO1.4particle selection
2EPUimage acquisition
4Gctf1.06CTF correction
7UCSF Chimera1.14model fitting
9PHENIX1.19.2model refinement
10cryoSPARC2.8initial Euler assignment
11RELION3.1final Euler assignment
13RELION3.13D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.3 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 149876 / Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT
Atomic model building
IDPDB-ID 3D fitting-IDAccession codeInitial refinement model-IDSource nameType
16F0L

6f0l

16F0L1PDBexperimental model
25BK4

5bk4

15BK42PDBexperimental model
36EYC

6eyc

16EYC3PDBexperimental model
46YA7

6ya7

16YA74PDBexperimental model
53QBZ

3qbz

13QBZ5PDBexperimental model
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 134.24 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.002568738
ELECTRON MICROSCOPYf_angle_d0.538993447
ELECTRON MICROSCOPYf_chiral_restr0.041710778
ELECTRON MICROSCOPYf_plane_restr0.004111647
ELECTRON MICROSCOPYf_dihedral_angle_d12.335610144

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