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 and Christian Speck (chris.speck@imperial.ac.uk)
Additional contact information
Jennifer N. Wells: Imperial College London
Lucy V. Edwardes: Imperial College London
Vera Leber: Imperial College London
Shenaz Allyjaun: Imperial College London
Matthew Peach: Imperial College London
Joshua Tomkins: Imperial College London
Antonia Kefala-Stavridi: Imperial College London
Sarah V. Faull: Imperial College London
Ricardo Aramayo: Imperial College London
Carolina M. Pestana: Imperial College London
Lepakshi Ranjha: Imperial College London
Christian Speck: Imperial College London

Nature Communications, 2025, vol. 16, issue 1, 1-21

Abstract: 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.

Date: 2025
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DOI: 10.1038/s41467-024-55772-z

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