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TitleReconstitution of human DNA licensing and the structural and functional analysis of key intermediates.
Journal, issue, pagesNat Commun, Vol. 16, Issue 1, Page 478, Year 2025
Publish dateJan 8, 2025
AuthorsJennifer 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 /
PubMed AbstractHuman 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.
External linksNat Commun / PubMed:39779677 / PubMed Central
MethodsEM (single particle)
Resolution6.68 Å
Structure data

19566

8rwv

EMDB-19566, PDB-8rwv:
Human OCCM DNA licensing intermediate
Method: EM (single particle) / Resolution: 6.68 Å

Source
  • homo sapiens (human)
KeywordsREPLICATION / DNA Replication / MCM2-7 / DNA licensing / human / origin / ORC / pre-RC / Cdc6 / Cdt1

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