Nature Communications (Jan 2025)

Reconstitution of human DNA licensing and the structural and functional analysis of key intermediates

  • 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

DOI
https://doi.org/10.1038/s41467-024-55772-z
Journal volume & issue
Vol. 16, no. 1
pp. 1 – 21

Abstract

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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 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.