Reconstitution of human DNA licensing and the structural and functional analysis of key intermediates
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 usi...
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Nature Portfolio
2025-01-01
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| Series: | Nature Communications |
| Online Access: | https://doi.org/10.1038/s41467-024-55772-z |
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| author | 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 |
| author_facet | 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 |
| author_sort | Jennifer N. Wells |
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| description | 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. |
| format | Article |
| id | doaj-art-2891be98d66d4bab851523e9c29c8626 |
| institution | Kabale University |
| issn | 2041-1723 |
| language | English |
| publishDate | 2025-01-01 |
| publisher | Nature Portfolio |
| record_format | Article |
| series | Nature Communications |
| spelling | doaj-art-2891be98d66d4bab851523e9c29c86262025-01-12T12:30:44ZengNature PortfolioNature Communications2041-17232025-01-0116112110.1038/s41467-024-55772-zReconstitution of human DNA licensing and the structural and functional analysis of key intermediatesJennifer N. Wells0Lucy V. Edwardes1Vera Leber2Shenaz Allyjaun3Matthew Peach4Joshua Tomkins5Antonia Kefala-Stavridi6Sarah V. Faull7Ricardo Aramayo8Carolina M. Pestana9Lepakshi Ranjha10Christian Speck11DNA Replication Group, Institute of Clinical Sciences, Faculty of Medicine, Imperial College LondonDNA Replication Group, Institute of Clinical Sciences, Faculty of Medicine, Imperial College LondonDNA Replication Group, Institute of Clinical Sciences, Faculty of Medicine, Imperial College LondonDNA Replication Group, Institute of Clinical Sciences, Faculty of Medicine, Imperial College LondonDNA Replication Group, Institute of Clinical Sciences, Faculty of Medicine, Imperial College LondonDNA Replication Group, Institute of Clinical Sciences, Faculty of Medicine, Imperial College LondonDNA Replication Group, Institute of Clinical Sciences, Faculty of Medicine, Imperial College LondonDNA Replication Group, Institute of Clinical Sciences, Faculty of Medicine, Imperial College LondonDNA Replication Group, Institute of Clinical Sciences, Faculty of Medicine, Imperial College LondonDNA Replication Group, Institute of Clinical Sciences, Faculty of Medicine, Imperial College LondonDNA Replication Group, Institute of Clinical Sciences, Faculty of Medicine, Imperial College LondonDNA Replication Group, Institute of Clinical Sciences, Faculty of Medicine, Imperial College LondonAbstract 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.https://doi.org/10.1038/s41467-024-55772-z |
| spellingShingle | 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 Reconstitution of human DNA licensing and the structural and functional analysis of key intermediates Nature Communications |
| title | Reconstitution of human DNA licensing and the structural and functional analysis of key intermediates |
| title_full | Reconstitution of human DNA licensing and the structural and functional analysis of key intermediates |
| title_fullStr | Reconstitution of human DNA licensing and the structural and functional analysis of key intermediates |
| title_full_unstemmed | Reconstitution of human DNA licensing and the structural and functional analysis of key intermediates |
| title_short | Reconstitution of human DNA licensing and the structural and functional analysis of key intermediates |
| title_sort | reconstitution of human dna licensing and the structural and functional analysis of key intermediates |
| url | https://doi.org/10.1038/s41467-024-55772-z |
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