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- PDB-7pt7: Structure of MCM2-7 DH complexed with Cdc7-Dbf4 in the presence o... -

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Basic information

Entry
Database: PDB / ID: 7pt7
TitleStructure of MCM2-7 DH complexed with Cdc7-Dbf4 in the presence of ADP:BeF3, state I
Components
  • (DNA replication licensing factor ...) x 5
  • Cell division control protein 7
  • DDK kinase regulatory subunit DBF4
  • Minichromosome maintenance protein 5
  • Undefined Mcm4 flexible N-terminal tail
KeywordsREPLICATION / Helicase / Activation / Kinase / Phosphorylation
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 DNA replication initiation / positive regulation of kinetochore assembly / positive regulation of meiotic DNA double-strand break formation / negative regulation of exit from mitosis / Dbf4-dependent protein kinase complex / positive regulation of protein localization to kinetochore / positive regulation of nuclear cell cycle DNA replication / positive regulation of meiosis I ...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 DNA replication initiation / positive regulation of kinetochore assembly / positive regulation of meiotic DNA double-strand break formation / negative regulation of exit from mitosis / Dbf4-dependent protein kinase complex / positive regulation of protein localization to kinetochore / positive regulation of nuclear cell cycle DNA replication / positive regulation of meiosis I / 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 / 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 / protein-containing complex localization / DNA replication preinitiation complex / MCM complex / replication fork protection complex / mitotic DNA replication checkpoint signaling / 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 / DNA strand elongation involved in DNA replication / nuclear replication fork / DNA replication origin binding / chromosome, centromeric region / DNA replication initiation / subtelomeric heterochromatin formation / protein serine/threonine kinase activator activity / helicase activity / chromosome segregation / transcription elongation by RNA polymerase II / 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 / eukaryotic translation initiation factor 2alpha kinase activity / chromosome, telomeric region / 3-phosphoinositide-dependent protein kinase activity / DNA-dependent protein kinase activity / ribosomal protein S6 kinase activity / histone H3S10 kinase activity / histone H2AXS139 kinase activity / histone H3S28 kinase activity / histone H4S1 kinase activity / histone H2BS14 kinase activity / histone H3T3 kinase activity / histone H2AS121 kinase activity / Rho-dependent protein serine/threonine kinase activity / histone H2BS36 kinase activity / histone H3S57 kinase activity / histone H2AT120 kinase activity / AMP-activated protein kinase activity / histone H2AS1 kinase activity / histone H3T6 kinase activity / histone H3T11 kinase activity / histone H3T45 kinase activity / DNA replication / non-specific serine/threonine protein kinase / cell division / protein serine kinase activity / protein serine/threonine kinase activity / centrosome / DNA damage response / 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 / 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 / BRCT domain superfamily / Serine/threonine-protein kinase, active site / Serine/Threonine protein kinases active-site signature. / Winged helix-like DNA-binding domain superfamily / Protein kinase domain / 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 / BERYLLIUM TRIFLUORIDE ION / 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)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.8 Å
AuthorsSaleh, A. / Noguchi, Y. / Aramayo, R. / Ivanova, M.E. / Speck, C.
Funding support United Kingdom, 4items
OrganizationGrant numberCountry
Biotechnology and Biological Sciences Research Council (BBSRC)BB/T005378/1 United Kingdom
Wellcome Trust107903/Z/15/Z United Kingdom
Medical Research Council (MRC, United Kingdom)A652-5PY40 United Kingdom
Wellcome Trust206175/Z/17/Z United Kingdom
CitationJournal: Nat Commun / Year: 2022
Title: The structural basis of Cdc7-Dbf4 kinase dependent targeting and phosphorylation of the MCM2-7 double hexamer.
Authors: Almutasem Saleh / Yasunori Noguchi / Ricardo Aramayo / Marina E Ivanova / Kathryn M Stevens / Alex Montoya / S Sunidhi / Nicolas Lopez Carranza / Marcin J Skwark / Christian Speck /
Abstract: The controlled assembly of replication forks is critical for genome stability. The Dbf4-dependent Cdc7 kinase (DDK) initiates replisome assembly by phosphorylating the MCM2-7 replicative helicase at ...The controlled assembly of replication forks is critical for genome stability. The Dbf4-dependent Cdc7 kinase (DDK) initiates replisome assembly by phosphorylating the MCM2-7 replicative helicase at the N-terminal tails of Mcm2, Mcm4 and Mcm6. At present, it remains poorly understood how DDK docks onto the helicase and how the kinase targets distal Mcm subunits for phosphorylation. Using cryo-electron microscopy and biochemical analysis we discovered that an interaction between the HBRCT domain of Dbf4 with Mcm2 serves as an anchoring point, which supports binding of DDK across the MCM2-7 double-hexamer interface and phosphorylation of Mcm4 on the opposite hexamer. Moreover, a rotation of DDK along its anchoring point allows phosphorylation of Mcm2 and Mcm6. In summary, our work provides fundamental insights into DDK structure, control and selective activation of the MCM2-7 helicase during DNA replication. Importantly, these insights can be exploited for development of novel DDK inhibitors.
History
DepositionSep 26, 2021Deposition site: PDBE / Processing site: PDBE
Revision 1.0Jun 8, 2022Provider: repository / Type: Initial release
Revision 1.1Jul 17, 2024Group: Data collection / Category: chem_comp_atom / chem_comp_bond / em_admin / Item: _em_admin.last_update

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
1: Undefined Mcm4 flexible N-terminal tail
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
8: Cell division control protein 7
9: DDK kinase regulatory subunit DBF4
B: DNA replication licensing factor MCM2
C: DNA replication licensing factor MCM3
D: DNA replication licensing factor MCM4
E: Minichromosome maintenance protein 5
F: DNA replication licensing factor MCM6
G: DNA replication licensing factor MCM7
hetero molecules


Theoretical massNumber of molelcules
Total (without water)1,359,45361
Polymers1,352,31215
Non-polymers7,14146
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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Protein/peptide , 1 types, 1 molecules 1

#1: Protein/peptide Undefined Mcm4 flexible N-terminal tail


Mass: 358.434 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 / Production host: Saccharomyces cerevisiae (brewer's yeast) / References: DNA helicase

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DNA replication licensing factor ... , 5 types, 10 molecules 2B3C4D6F7G

#2: 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 (brewer's yeast) / References: UniProt: P29469, DNA helicase
#3: Protein DNA replication licensing factor MCM3 / Minichromosome maintenance protein 3


Mass: 107653.508 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 (brewer's yeast) / References: UniProt: P24279, DNA helicase
#4: 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 (brewer's yeast) / References: UniProt: P30665, DNA helicase
#6: 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 (brewer's yeast) / References: UniProt: P53091, DNA helicase
#7: 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 (brewer's yeast) / References: UniProt: P38132, DNA helicase

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Protein , 3 types, 4 molecules 5E89

#5: 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 (brewer's yeast) / References: UniProt: P29496, DNA helicase
#8: 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 (brewer's yeast)
References: UniProt: P06243, non-specific serine/threonine protein kinase
#9: Protein DDK kinase regulatory subunit DBF4 / Dumbbell forming protein 4


Mass: 80824.156 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 (brewer's yeast) / References: UniProt: P32325

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Non-polymers , 4 types, 46 molecules

#10: Chemical
ChemComp-ADP / ADENOSINE-5'-DIPHOSPHATE


Mass: 427.201 Da / Num. of mol.: 13 / Source method: obtained synthetically / Formula: C10H15N5O10P2 / Feature type: SUBJECT OF INVESTIGATION / Comment: ADP, energy-carrying molecule*YM
#11: Chemical
ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 14 / Source method: obtained synthetically / Formula: Mg / Feature type: SUBJECT OF INVESTIGATION
#12: Chemical
ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 12 / Source method: obtained synthetically / Formula: Zn / Feature type: SUBJECT OF INVESTIGATION
#13: Chemical
ChemComp-BEF / BERYLLIUM TRIFLUORIDE ION


Mass: 66.007 Da / Num. of mol.: 7 / Source method: obtained synthetically / Formula: BeF3 / Feature type: SUBJECT OF INVESTIGATION

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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: MCM2-7 double hexamer bound to one copy of Cdc7-Dbf4 / Type: COMPLEX / Entity ID: #1-#9 / Source: RECOMBINANT
Molecular weightValue: 1.35 MDa / Experimental value: NO
Source (natural)Organism: Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Source (recombinant)Organism: Saccharomyces cerevisiae (brewer's yeast)
Buffer solutionpH: 7.5
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: 15 mA / Grid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R2/2
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 K / Details: blot for 1.5 seconds and blot force +2

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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 / Cs: 2.7 mm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 48.4 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) / Num. of real images: 13470

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Processing

EM software
IDNameCategory
2EPUimage acquisition
4CTFFINDCTF correction
10RELIONinitial Euler assignment
11RELIONfinal Euler assignment
12RELIONclassification
13RELION3D reconstruction
14PHENIX3D reconstruction
CTF correctionType: PHASE FLIPPING ONLY
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.8 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 30807
Details: Multi-body auto-refined maps were combined to generate a composite map using an atomic model (derived from MD-(ADP:BeF3) state I), with the program combine_focused_maps in Phenix
Symmetry type: POINT

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