- EMDB-19566: Human OCCM DNA licensing intermediate -
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Basic information
Entry
Database: EMDB / ID: EMD-19566
Title
Human OCCM DNA licensing intermediate
Map data
Sample
Complex: hOCCM
Protein or peptide: x 12 types
DNA: x 2 types
Keywords
DNA Replication / MCM2-7 / DNA licensing / human / origin / ORC / pre-RC / Cdc6 / Cdt1 / REPLICATION
Function / homology
Function and homology information
DNA replication preinitiation complex assembly / response to sorbitol / positive regulation of chromosome segregation / positive regulation of chromatin binding / 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 ...DNA replication preinitiation complex assembly / response to sorbitol / positive regulation of chromosome segregation / positive regulation of chromatin binding / 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 / Regulation of MITF-M-dependent genes involved in DNA replication, damage repair and senescence / alpha DNA polymerase:primase complex / mitotic DNA replication / regulation of phosphorylation / DNA replication checkpoint signaling / attachment of mitotic spindle microtubules to kinetochore / CMG complex / 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 / Transcription of E2F targets under negative control by DREAM complex / neural precursor cell proliferation / regulation of mitotic metaphase/anaphase transition / regulation of DNA-templated DNA replication initiation / DNA strand elongation involved in DNA replication / positive regulation of cytokinesis / negative regulation of DNA replication / spindle midzone / G1/S-Specific Transcription / negative regulation of cell cycle / regulation of DNA replication / cellular response to angiotensin / DNA replication origin binding / cochlea development / protein polymerization / Activation of the pre-replicative complex / DNA replication initiation / 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 / Assembly of the ORC complex at the origin of replication / protein serine/threonine kinase binding / cellular response to epidermal growth factor stimulus / Assembly of the pre-replicative complex / positive regulation of DNA replication / CDK-mediated phosphorylation and removal of Cdc6 / helicase activity / kinetochore / Orc1 removal from chromatin / cellular response to xenobiotic stimulus / positive regulation of fibroblast proliferation / spindle pole / mitotic spindle / mitotic cell cycle / nucleosome assembly / single-stranded DNA binding / histone binding / DNA helicase / chromosome, telomeric region / DNA replication / cell population proliferation / nuclear body / cilium / negative regulation of cell population proliferation / cell division / nucleotide binding / intracellular membrane-bounded organelle / apoptotic process / centrosome / DNA damage response / 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
Journal: 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.
Film or detector model: GATAN K3 (6k x 4k) / Digitization - Dimensions - Width: 5760 pixel / Digitization - Dimensions - Height: 4092 pixel / Number grids imaged: 1 / Number real images: 18022 / Average exposure time: 3.0 sec. / Average electron dose: 40.0 e/Å2
Electron beam
Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Type of model: OTHER Details: AlphaFold models from each individual subunit were rigid body docked into the experimental map before model building in COOT.
Final reconstruction
Number classes used: 1 / Resolution.type: BY AUTHOR / Resolution: 6.68 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 8730
Initial angle assignment
Type: MAXIMUM LIKELIHOOD
Final angle assignment
Type: MAXIMUM LIKELIHOOD
FSC plot (resolution estimation)
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