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Mechanisms for licensing origins of DNA replication in eukaryotic cells

Nature Structural & Molecular Biology volume 32, pages 1143–1153 (2025)Cite this article

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Abstract

The initiation of DNA replication in eukaryotic cells begins with the assembly of pre-replicative complexes (pre-RCs) at many sites along each chromosome during the G1 phase of the cell cycle. Pre-RCs license each chromosome for duplication during S phase and mark the origins of DNA replication. In this Review, we discuss and contextualize recent findings identifying the mechanisms of origin recognition and pre-RC assembly mediated by the origin recognition complex (ORC), Cdc6 and the Mcm2–Mcm7 (Mcm2-7) hexamer bound to Cdt1. We also present comprehensive videos that demonstrate the multiple mechanisms for pre-RC assembly and compare the structures of the complexes involved in human and Saccharomyces cerevisiae cells.

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Fig. 1: A general scheme for pre-RC assembly ensuring faithful duplication of all chromosomes.
Fig. 2: The assembly of a pre-RC with one ORC loading the Mcm2-7 double hexamer.
Fig. 3: Dynamic structure of the ORC.
Fig. 4: The process of establishment of pre-RCs at an S. cerevisiae origin in which the B2 domain is separated from the A and B1 origin elements.
Fig. 5: The MCM double hexamer bound to origin DNA.

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Acknowledgements

The studies of DNA replication in the author’s laboratories have been supported by grants: NIH grant GM45346, the Goldring Family Foundation and Cold Spring Harbor Laboratory (to B.S.); the Francis Crick Institute, which receives its core funding from Cancer Research UK (CC2002), the UK Medical Research Council (CC2002) and the Wellcome Trust (CC2002); Wellcome Trust Senior Investigator Awards (106252/Z/14/Z and 219527/Z/19/Z); and European Research Council Advanced Grants (669424-CHROMOREP and 101020432-MeChroRep) to J.F.X.D. Research on animation in cell biology has been supported by NIH grant U54 AI170856 (to J.H.I.). The production of the videos in this Review was supported by a grant to J.H.I. from Cold Spring Harbor Laboratory.

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Authors and Affiliations

  1. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, USA

    Bruce Stillman

  2. Chromosome Replication Laboratory, The Francis Crick Institute, London, UK

    John F. X. Diffley

  3. Department of Biochemistry, University of Utah, Salt Lake City, UT, USA

    Janet H. Iwasa

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  1. Bruce Stillman
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Correspondence to Bruce Stillman.

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Nature Structural & Molecular Biology thanks David MacAlpine, Yuanliang Zhai, and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Dimitris Typas was the primary editor on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.

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Supplementary information

Supplementary Video 1

The process of establishing pre-RCs at the S. cerevisiae ARS1 origin by a single ORC that cooperates with Cdc6 and Cdt1 to load two Mcm2-7 (MCM) hexamers in a head-to-head manner at each origin. A positioned nucleosome is bound to the DNA adjacent to the ARS1 origin. The A/B1 ORC-binding site is relatively close to the inverted but weaker ORC-binding site B2. One ORC binds first to the A and B1 element and, with Cdc6 and Cdt1, loads the first MCM hexamer and then flips to the B2 element, where it cooperates with new Cdc6 and Cdt1 proteins to load a second MCM hexamer that then rotates to form the MCM double hexamer. The MCM hexamer bound to Cdt1 interacts with ORC–Cdc6 on DNA through WH domains in the MCM hexamer (initially in the Mcm3 and Mcm7 subunits of MCM (highlighted purple domains)). The source of the structures for this movie is the same as those in the Figure 2 legend. The structures of pre-RC components, DNA and nucleosomes were downloaded from the RCSB PDB (http://rcsb.org) and used for animation and as structural references (PDB IDs: 5V8F, 5XF8, 5BK4, 5ZR1, 6OM3, 6RQC, 6WGG, 7MCA, 5UJ7, 7JPO, 7JPP, 7JPQ, 7JPR, 7JPS and 1AOI). Intrinsically disordered regions that were missing from published structures were modeled using AlphaFold (https://alphafold.ebi.ac.uk/). Molecular structures were exported as meshes using UCSF Chimera (https://www.cgl.ucsf.edu/chimera/) and animation of molecular processes was completed using Autodesk Maya.

Supplementary Video 2

A comparison of the structures of the S. cerevisiae ORC and the human ORC. The human ORC can exist in an autoinhibited structure with the ORC1 subunit far away from the ORC4 subunit and the ORC2 winged helix in the channel that binds DNA. The ORC1 subunit rotates to interact with ORC4 to create an active ATP-binding pocket and the ORC2 WH domain moves out of the DNA channel to allow ORC to bind DNA. The sources of the structures were PDB IDs: 5ZR1 (yeast ORC), 5UJ7 (human ORC), 7JPO, 7JPP, 7JPQ, 7JPR and 7JPS (human ORC).

Supplementary Video 3

The process of establishment of pre-RCs at S. cerevisiae origins of DNA replication, where the inverted B2 ORC binding site is further apart from the A/B1 element. In this case, the first MCM hexamer bound to Cdt1 is loaded by an ORC-–dc6 complex and then the second MCM hexamer bound to Cdt1 is loaded by a second ORC–Cdc6 complex. The MCM hexamers rotate along the DNA, forming the MCM double hexamer. The annotation is similar to the annotation in Supplementary Video 1. The sources of the structures are the same as shown in the Figure 2 legend.

Supplementary Video 4

Comparison of the S. cerevisiae MCM double hexamer and the human MCM double hexamer. The DNA in the S. cerevisiae double hexamer has duplex DNA pass through the MCM proteins in a zig-zag manner but is not unwound. The human MCM double hexamer is similar to the S. cerevisiae MCM double hexamer but the DNA in the human MCM double hexamer is unwound and the bases in the central base pair are completely separated. The sources of the structures were PDBs 5BK4 (yeast MCM double hexamer), 7W1Y (human MCM double hexamer).

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Stillman, B., Diffley, J.F.X. & Iwasa, J.H. Mechanisms for licensing origins of DNA replication in eukaryotic cells. Nat Struct Mol Biol 32, 1143–1153 (2025). https://doi.org/10.1038/s41594-025-01587-5

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