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- PDB-8rwv: Human OCCM DNA licensing intermediate -

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Basic information

Entry
Database: PDB / ID: 8rwv
TitleHuman OCCM DNA licensing intermediate
Components
  • (DNA replication licensing factor ...) x 4
  • (DNA) x 2
  • (Isoform 2 of ...) x 2
  • (Origin recognition complex subunit ...) x 4
  • Cell division control protein 6 homolog
  • DNA replication factor Cdt1
KeywordsREPLICATION / DNA Replication / MCM2-7 / DNA licensing / human / origin / ORC / pre-RC / Cdc6 / Cdt1
Function / homology
Function and homology information


DNA replication preinitiation complex assembly / response to sorbitol / positive regulation of chromosome segregation / cellular response to vasopressin / polar body extrusion after meiotic divisions / CDC6 association with the ORC:origin complex / origin recognition complex / positive regulation of DNA-templated DNA replication / regulation of nuclear cell cycle DNA replication / E2F-enabled inhibition of pre-replication complex formation ...DNA replication preinitiation complex assembly / response to sorbitol / positive regulation of chromosome segregation / cellular response to vasopressin / polar body extrusion after meiotic divisions / CDC6 association with the ORC:origin complex / origin recognition complex / positive regulation of DNA-templated DNA replication / regulation of nuclear cell cycle DNA replication / E2F-enabled inhibition of pre-replication complex formation / Switching of origins to a post-replicative state / Unwinding of DNA / negative regulation of DNA-templated DNA replication / nuclear origin of replication recognition complex / traversing start control point of mitotic cell cycle / alpha DNA polymerase:primase complex / Regulation of MITF-M-dependent genes involved in DNA replication, damage repair and senescence / mitotic DNA replication / CMG complex / attachment of mitotic spindle microtubules to kinetochore / DNA replication checkpoint signaling / regulation of phosphorylation / inner kinetochore / nuclear pre-replicative complex / DNA replication preinitiation complex / MCM complex / regulation of cyclin-dependent protein serine/threonine kinase activity / mitotic DNA replication checkpoint signaling / double-strand break repair via break-induced replication / mitotic DNA replication initiation / positive regulation of chromatin binding / Transcription of E2F targets under negative control by DREAM complex / regulation of DNA-templated DNA replication initiation / neural precursor cell proliferation / regulation of mitotic metaphase/anaphase transition / DNA strand elongation involved in DNA replication / positive regulation of cytokinesis / spindle midzone / G1/S-Specific Transcription / negative regulation of DNA replication / negative regulation of cell cycle / regulation of DNA replication / cellular response to angiotensin / DNA replication origin binding / cochlea development / protein polymerization / DNA replication initiation / Activation of the pre-replicative complex / glial cell proliferation / Activation of ATR in response to replication stress / heterochromatin / intercellular bridge / DNA polymerase binding / cellular response to interleukin-4 / DNA helicase activity / protein serine/threonine kinase binding / Assembly of the ORC complex at the origin of replication / cellular response to epidermal growth factor stimulus / positive regulation of DNA replication / Assembly of the pre-replicative complex / helicase activity / kinetochore / CDK-mediated phosphorylation and removal of Cdc6 / cellular response to xenobiotic stimulus / positive regulation of fibroblast proliferation / Orc1 removal from chromatin / spindle pole / mitotic spindle / mitotic cell cycle / nucleosome assembly / single-stranded DNA binding / histone binding / DNA helicase / DNA replication / chromosome, telomeric region / cell population proliferation / nuclear body / cilium / negative regulation of cell population proliferation / cell division / nucleotide binding / intracellular membrane-bounded organelle / apoptotic process / DNA damage response / centrosome / chromatin binding / chromatin / nucleolus / perinuclear region of cytoplasm / enzyme binding / negative regulation of transcription by RNA polymerase II / ATP hydrolysis activity / DNA binding / zinc ion binding / nucleoplasm / ATP binding / metal ion binding / identical protein binding / nucleus / membrane
Similarity search - Function
CDT1 Geminin-binding domain-like / DNA replication factor Cdt1 / DNA replication factor CDT1 like / DNA replication factor CDT1 like / DNA replication factor Cdt1, C-terminal / DNA replication factor Cdt1, C-terminal WH domain superfamily / DNA replication factor Cdt1 C-terminal domain / Cell division protein Cdc6/18 / : / Cdc6/ORC-like, ATPase lid domain ...CDT1 Geminin-binding domain-like / DNA replication factor Cdt1 / DNA replication factor CDT1 like / DNA replication factor CDT1 like / DNA replication factor Cdt1, C-terminal / DNA replication factor Cdt1, C-terminal WH domain superfamily / DNA replication factor Cdt1 C-terminal domain / Cell division protein Cdc6/18 / : / Cdc6/ORC-like, ATPase lid domain / Origin recognition complex subunit 3, insertion domain / Origin recognition complex subunit 3 insertion domain / CDC6, C terminal / Cdc6, C-terminal / CDC6, C terminal winged helix domain / Origin recognition complex subunit 4 / Origin recognition complex, subunit 3 / Origin recognition complex, subunit 5 / Origin recognition complex subunit 4, C-terminal / Origin recognition complex subunit 3, winged helix C-terminal / Origin recognition complex subunit 3, N-terminal / : / : / Origin recognition complex (ORC) subunit 3 N-terminus / Origin recognition complex (ORC) subunit 4 C-terminus / Origin recognition complex (ORC) subunit 5 C-terminus / Origin recognition complex winged helix C-terminal / ORC5, lid domain / Orc1-like, AAA ATPase domain / : / : / Origin recognition complex subunit 2 RecA-like domain / ORC2 WHD / AAA ATPase domain / Origin recognition complex, subunit 2 / DNA replication licensing factor MCM2-like, winged-helix domain / AAA lid domain / AAA lid domain / : / DNA replication licensing factor MCM7, winged helix / 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 / Bromo adjacent homology domain / BAH domain / Bromo adjacent homology (BAH) domain / Bromo adjacent homology (BAH) domain superfamily / BAH domain profile. / 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 / ATPase family associated with various cellular activities (AAA) / ATPase, AAA-type, core / Winged helix DNA-binding domain superfamily / 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
DNA / DNA (> 10) / Origin recognition complex subunit 5 / Origin recognition complex subunit 4 / DNA replication licensing factor MCM3 / DNA replication licensing factor MCM4 / DNA replication licensing factor MCM7 / DNA replication licensing factor MCM2 / Origin recognition complex subunit 1 / Origin recognition complex subunit 2 ...DNA / DNA (> 10) / Origin recognition complex subunit 5 / Origin recognition complex subunit 4 / DNA replication licensing factor MCM3 / DNA replication licensing factor MCM4 / DNA replication licensing factor MCM7 / DNA replication licensing factor MCM2 / Origin recognition complex subunit 1 / Origin recognition complex subunit 2 / DNA replication licensing factor MCM6 / Cell division control protein 6 homolog / DNA replication factor Cdt1 / Origin recognition complex subunit 3
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 6.68 Å
AuthorsWells, J.N. / Leber, V. / Edwards, L.V. / Allyjaun, S. / Peach, M. / Tomkins, J. / Kefala-Stavridi, A. / Faull, S.V. / Aramayo, R. / Pestana, C.M. ...Wells, J.N. / Leber, V. / Edwards, L.V. / Allyjaun, S. / Peach, M. / Tomkins, J. / Kefala-Stavridi, A. / Faull, S.V. / Aramayo, R. / Pestana, C.M. / Ranjha, L. / Speck, C.
Funding support United Kingdom, 1items
OrganizationGrant numberCountry
Cancer Research UK100006 United Kingdom
CitationJournal: Nat Commun / Year: 2025
Title: Reconstitution of human DNA licensing and the structural and functional analysis of key intermediates.
Authors: Jennifer N Wells / Lucy V Edwardes / Vera Leber / Shenaz Allyjaun / Matthew Peach / Joshua Tomkins / Antonia Kefala-Stavridi / Sarah V Faull / Ricardo Aramayo / Carolina M Pestana / Lepakshi ...Authors: Jennifer N Wells / Lucy V Edwardes / Vera Leber / Shenaz Allyjaun / Matthew Peach / Joshua Tomkins / Antonia Kefala-Stavridi / Sarah V Faull / Ricardo Aramayo / Carolina M Pestana / Lepakshi Ranjha / Christian Speck /
Abstract: Human DNA licensing initiates replication fork assembly and DNA replication. This reaction promotes the loading of the hMCM2-7 complex on DNA, which represents the core of the replicative helicase ...Human DNA licensing initiates replication fork assembly and DNA replication. This reaction promotes the loading of the hMCM2-7 complex on DNA, which represents the core of the replicative helicase that unwinds DNA during S-phase. Here, we report the reconstitution of human DNA licensing using purified proteins. We showed that the in vitro reaction is specific and results in the assembly of high-salt resistant hMCM2-7 double-hexamers. With ATPγS, an hORC1-5-hCDC6-hCDT1-hMCM2-7 (hOCCM) assembles independent of hORC6, but hORC6 enhances double-hexamer formation. We determined the hOCCM structure, which showed that hORC-hCDC6 recruits hMCM2-7 via five hMCM winged-helix domains. The structure highlights how hORC1 activates the hCDC6 ATPase and uncovered an unexpected role for hCDC6 ATPase in complex disassembly. We identified that hCDC6 binding to hORC1-5 stabilises hORC2-DNA interactions and supports hMCM3-dependent recruitment of hMCM2-7. Finally, the structure allowed us to locate cancer-associated mutations at the hCDC6-hMCM3 interface, which showed specific helicase loading defects.
History
DepositionFeb 5, 2024Deposition site: PDBE / Processing site: PDBE
Revision 1.0Feb 5, 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
6: DNA replication licensing factor MCM6
7: DNA replication licensing factor MCM7
A: Origin recognition complex subunit 1
B: Origin recognition complex subunit 2
C: Isoform 2 of Origin recognition complex subunit 3
D: Origin recognition complex subunit 4
E: Origin recognition complex subunit 5
F: Cell division control protein 6 homolog
G: DNA replication factor Cdt1
H: DNA
J: DNA


Theoretical massNumber of molelcules
Total (without water)967,37414
Polymers967,37414
Non-polymers00
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 licensing factor ... , 4 types, 4 molecules 2467

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


Mass: 106435.773 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: Homo sapiens (human) / 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: Homo sapiens (human) / References: UniProt: P33991, DNA helicase
#4: 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: Homo sapiens (human) / References: UniProt: Q14566, DNA helicase
#5: 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: Homo sapiens (human) / References: UniProt: P33993, DNA helicase

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Isoform 2 of ... , 2 types, 2 molecules 3C

#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: Homo sapiens (human) / References: UniProt: P25205, DNA helicase
#8: Protein Isoform 2 of Origin recognition complex subunit 3 / Origin recognition complex subunit Latheo


Mass: 82436.133 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: ORC3, LATHEO, ORC3L / Production host: Saccharomyces cerevisiae S288C (yeast) / References: UniProt: Q9UBD5

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Origin recognition complex subunit ... , 4 types, 4 molecules ABDE

#6: Protein Origin recognition complex subunit 1 / Replication control protein 1


Mass: 108813.562 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: ORC1, ORC1L, PARC1 / Production host: Saccharomyces cerevisiae S288C (yeast) / References: UniProt: Q13415
#7: Protein Origin recognition complex subunit 2


Mass: 66063.375 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: ORC2, ORC2L / Production host: Saccharomyces cerevisiae S288C (yeast) / References: UniProt: Q13416
#9: Protein Origin recognition complex subunit 4


Mass: 50443.266 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: ORC4, ORC4L / Production host: Saccharomyces cerevisiae S288C (yeast) / References: UniProt: O43929
#10: Protein Origin recognition complex subunit 5


Mass: 50349.934 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: ORC5, ORC5L / Production host: Saccharomyces cerevisiae S288C (yeast) / References: UniProt: O43913

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Protein , 2 types, 2 molecules FG

#11: Protein Cell division control protein 6 homolog / CDC6-related protein / Cdc18-related protein / HsCdc18 / p62(cdc6) / HsCDC6


Mass: 62834.379 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: CDC6, CDC18L / Production host: Escherichia coli (E. coli) / References: UniProt: Q99741
#12: Protein DNA replication factor Cdt1 / Double parked homolog / DUP


Mass: 46099.020 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: CDT1 / Production host: Escherichia coli (E. coli) / References: UniProt: Q9H211

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DNA chain , 2 types, 2 molecules HJ

#13: DNA chain DNA


Mass: 11933.735 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Production host: Homo sapiens (human)
#14: DNA chain DNA


Mass: 12379.996 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Production host: Homo sapiens (human)

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Details

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: hOCCM / Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Molecular weightValue: 1.01 MDa / Experimental value: NO
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Homo sapiens (human)
Buffer solutionpH: 7.5
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R2/2
VitrificationCryogen name: ETHANE

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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 defocus max: 2500 nm / Nominal defocus min: 500 nm / C2 aperture diameter: 50 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN
Image recordingAverage exposure time: 3 sec. / Electron dose: 40 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 18022
Image scansWidth: 5760 / Height: 4092

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Processing

EM softwareName: PHENIX / Version: 1.20.1_4487: / Category: model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 1598732
3D reconstructionResolution: 6.68 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 8730 / Num. of class averages: 1 / Symmetry type: POINT

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