2025-08-21_153057_nature_s41586-024-08237-8

Multiple mechanisms for licensing human replication origins | Nature

URL: https://www.nature.com/articles/s41586-024-08237-8

Saved: 2025-08-21T22:31:00.716Z

Article Published: 27 November 2024 Multiple mechanisms for licensing human replication origins Ran Yang, Olivia Hunker, Marleigh Wise & Franziska Bleichert

Nature volume 636, pages 488–498 (2024)Cite this article

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Abstract

Loading of replicative helicases is obligatory for the assembly of DNA replication machineries. The eukaryotic MCM2–7 replicative helicase motor is deposited onto DNA by the origin recognition complex (ORC) and co-loader proteins as a head-to-head double hexamer to license replication origins. Although extensively studied in budding yeast1,2,3,4, the mechanisms of origin licensing in multicellular eukaryotes remain poorly defined. Here we use biochemical reconstitution and electron microscopy to reconstruct the human MCM loading pathway. We find that unlike in yeast, the ORC6 subunit of the ORC is not essential for—but enhances—human MCM loading. Electron microscopy analyses identify several intermediates en route to MCM double hexamer formation in the presence and absence of ORC6, including a DNA-loaded, closed-ring MCM single hexamer intermediate that can mature into a head-to-head double hexamer through multiple mechanisms. ORC6 and ORC3 facilitate the recruitment of the ORC to the dimerization interface of the first hexamer into MCM–ORC (MO) complexes that are distinct from the yeast MO complex5,6 and may orient the ORC for second MCM hexamer loading. Additionally, MCM double hexamer formation can proceed through dimerization of independently loaded MCM single hexamers, promoted by a propensity of human MCM2–7 hexamers to self-dimerize. This flexibility in human MCM loading may provide resilience against cellular replication stress, and the reconstitution system will enable studies addressing outstanding questions regarding DNA replication initiation and replication-coupled events in the future.

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Additional access options: Log in Learn about institutional subscriptions Read our FAQs Contact customer support Similar content being viewed by others MCM double hexamer loading visualized with human proteins Article Open access 27 November 2024 DNA replication origins retain mobile licensing proteins Article Open access 26 March 2021 MCM2-7 loading-dependent ORC release ensures genome-wide origin licensing Article Open access 24 August 2024 Data availability

The PDB coordinates and cryo-EM maps have been deposited into the Protein Data Bank and Electron Microscopy Data Bank under the following accession numbers: PDB 8W0E and EMD-43707 for the loaded human MCM2–7 single hexamer, PDB 8W0F and EMD-43708 for the loaded human MCM2–7 double hexamer, PDB 8W0G and EMD-43709 for human MCM2–7 dimers, PDB 8W0I and EMD-43710 for the locally refined map containing one copy of the human MCM2–7 hexamer from the dimer, and PDB 9CAQ and EMD-45400 for human MCM2–7 double hexamers formed by dimerization of independently loaded MCM single hexamers without ORC6. The previously published model of human MCM-DH and the human ORC used for initial model building and docking are available in the Protein Data Bank using accession codes 7W1Y and 7JPO, respectively. The coordinates and cryo-EM maps of the S. cerevisiae MO can be accessed using accession codes PDB 6RQC and EMD-4980. S. cerevisiae coordinates for pre-insertion OCCM and OCCM are at PDB 6WGG and PDB 5V8F. Source data are provided with this paper.

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Acknowledgements

Cryo-EM data were collected at the Yale Cryo-EM resource and at the Laboratory for BioMolecular Structure (LBMS). The authors thank S. Wu, J. Lin, L. Wang, G. Hu and J. Kaminsky for assistance with microscope operation and data collection; H. Marzano for help with initial model building of the MCM dimer; and M. Hochstrasser, M. Solomon and members of the Bleichert laboratory for critical reading of the manuscript. This work is supported by the National Institutes of General Medicine (R01-GM141313 to F.B.). O.H. is supported by the NIH predoctoral programme in Biophysics (T32-GM008283) and the National Cancer Institute (1F31-CA278331-01). LBMS is supported by the DOE Office of Biological and Environmental Research (KP1607011). The Yale Cryo-EM Resource is funded in part by NIH grant S10OD023603.

Author information Author notes

These authors contributed equally: Ran Yang, Olivia Hunker

Authors and Affiliations

Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT, USA

Ran Yang, Olivia Hunker, Marleigh Wise & Franziska Bleichert

Contributions

F.B. conceptualized and supervised the project. R.Y., O.H., M.W. and F.B. cloned expression constructs and purified recombinant proteins. R.Y. and O.H. prepared samples and collected negative-stain EM and cryo-EM data. R.Y., O.H. and F.B. performed biochemical experiments, processed EM data, built and refined atomic models and wrote the manuscript.

Corresponding author

Correspondence to Franziska Bleichert.

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The authors declare no competing interests.

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Nature thanks the anonymous reviewer(s) for their contribution to the peer review of this work.

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Extended data figures and tables Extended Data Fig. 1 In vitro reconstitution of human origin licensing and validation of fluorescence-based MCM loading assay.

a) Electron micrograph of negatively stained particles from high-salt wash eluate of loading reactions with ATP. White arrows mark MCM double hexamers. b) N-terminal, intrinsically disordered regions (IDRs) in ORC1, CDC6, and CDT1 are not essential for human MCM loading. Silver-stained SDS-PAGE gels for bead-based MCM recruitment and loading assays are shown. N-terminally truncated ORC1 and CDT1 were used in all subsequent experiments unless noted otherwise. FL, full-length; Δ, truncated. c and d) Geminin inhibits MCM recruitment and loading in the in vitro reconstituted system. c) Silver-stained SDS-PAGE gels of inputs, recruited, and loaded proteins in the absence or presence of increasing concentrations of Geminin (labeled as molar fold excess compared to CDT1). Note that Geminin associates nonspecifically with beads in low-salt conditions. Full-length ORC and CDT1 were used in this experiment. d) Quantification of results in c. Means and standard deviations of MCM2 band intensities from three independent experiments normalized to the reaction without Geminin on each gel are plotted. e to h) Validation of fluorescence-based MCM loading assay. e) MCM2-7 with GFP fused to the C-terminus of MCM2 supports MCM recruitment and loading to similar levels as untagged (UT) MCM. Silver-stained SDS-PAGE gel is shown. f) Loaded MCM-GFP are double hexamers like untagged MCM2-7 as seen in negative-stain EM images of particles eluted from beads. g) Raw GFP fluorescence intensities in inputs and elutions from recruitment and loading reactions with MCM-GFP. h) The fluorescence intensity signal linearly increases with MCM-GFP concentration. Means and standard deviations from six independent experiments are plotted in g and h. a.u., arbitrary units. For gel source data, see Supplementary Information.

Source data

Extended Data Fig. 2 Full-length ORC6 facilitates while ORC1 is essential for human MCM loading.

a) Silver-stained SDS-PAGE gels of inputs and elutions from bead-based recruitment and loading reactions with and without ORC1. b) Silver-stained SDS-PAGE gels of elutions from bead-based recruitment and loading reactions with full-length human loading factors with or without ORC6. c to g) All ORC6 domains are required in cis to enhance human MCM loading. c) Domain architecture of human (Hs) ORC6. Numbers denote amino acid positions of domain boundaries. Truncated HsORC6 constructs are schematized below. d) SDS-PAGE gel of purified full-length (FL) wild-type (WT), truncated, and mutant ORC6 proteins used in this study. e) Experimental setup for assessing the contributions of ORC6 domains to MCM loading. f) MCM loading but not recruitment is impaired when ORC6 is absent or truncated. Means and standard deviations of MCM-GFP fluorescence in elutions of bead-based recruitment and loading assays (normalized to the average signal obtained with full-length ORC6) from three independent experiments are plotted. Dashed lines mark background signal in reactions without nucleotide. g) Quantification of MCM-DHs observed by EM in elutions of loading reactions (after high-salt wash). The numbers of double hexamers per micrograph were counted manually from three independent experiments, with 10 micrographs recorded for each sample per experimental repeat (total n = 30 micrographs per sample), and normalized to the +ORC6-FL sample. Black lines represent means. Statistical significance was determined using two-way ANOVA analysis and the Tukey’s multiple comparisons test (****P < 0.0001). Differences in MCM loading efficiencies without ORC6 and with ORC6 truncations between f and g are likely due to the presence of loaded MCM single hexamers that contribute to the signal measured in the fluorescence-based assay, thus slightly overestimating MCM loading efficiency. For gel source data, see Supplementary Information.

Source data

Extended Data Fig. 3 The human MO-like complex adopts different conformational states that are architecturally distinct from the S. cerevisiae (Sc) MO.

a) Class averages (negative stain) of purified human MCM2-7 or of loading reactions kept on ice. b) Quantification of MCM-containing assemblies (by negative-stain EM and 2D classification) in loading reactions without DNA. c) Projections of the ScMO cryo-EM map (EMD-49805, low-pass filtered to 20 Å). d) Negative-stain EM reconstruction of human MO-like complex state I, with structures of human MCM-SH (this study or PDB 7W1Y26) and human ORC (PDB 7JPO64) docked into the EM map. e) Superposition of MCMs in ScMO and human MO-like complex state I. The ORC6-binding regions in the ORC3 inserts are marked for reference. f) Low-pass filtered (to 15 Å) cryo-EM map of ScMO (EMD-4980 with PDB 6RQC5). g–h) The MO-like complex peaks early during MCM loading. g) Experimental workflow. h) Quantification of MCM-containing assemblies. i) Negative-stain EM reconstruction of human MO-like complex state II with MCM and ORC docked (as in d). j) Superposition of MCMs in ScMO and human MO-like complex state II. k–l) The ORC3 insert is positioned closer to the MCM2-NTD in MO state II (in k) than in MO state I (in l). Dashed lines in l indicate distances between consecutive amino acids if the ORC3 tether were to remain bound to MCM2. This interaction site is delineated by a dashed box in i. m) Deletion of the ORC3 tether sustains efficient MCM recruitment to DNA. Means and standard deviations of MCM-GFP fluorescence in bead-based recruitment assays from three independent experiments, normalized to the average of ORC3(WT)/+ORC6 reactions at each ORC/CDC6 concentration. n) Negative-stain electron micrograph region of MCM-DHs (white arrows) eluted from beads after high-salt wash from loading reactions with ORC3(ΔT). In b and h, two independent experiments with 100-150 micrographs each were analyzed per condition.

Source data

Extended Data Fig. 4 Cryo-EM data processing and validation of loaded human MCM2-7.

a) Cryo-EM image and b) 2D cryo-class averages from human MCM loading reactions. c) Cryo-EM data processing workflow for MCM single and double hexamer reconstructions. d and e) Angular distribution plots for reconstructed cryo-EM volumes of loaded MCM single (in d) and double (in e) hexamers.

Extended Data Fig. 5 Resolution estimation of loaded human MCM-SH and MCM-DH cryo-EM reconstructions.

a) Surface and cut-through views of unsharpened cryo-EM map of loaded MCM single hexamer. b) 3D Fourier shell correlation (FSC) plot for loaded MCM single hexamer reconstruction. c) Surface and cut-through views of unsharpened cryo-EM map of loaded MCM double hexamer. d) 3D FSC plot for loaded MCM double hexamer reconstruction. e) FSC curves of cryo-EM maps and refined models. f) Interior hairpin loop interactions with DNA are similar in MCM-DH and MCM-SH. Helix-2-insert (H2I), pre-sensor 1 β hairpin (PS1), and β-turn loops in each subunit are shown as cartoon and DNA is shown as surface. The β-turn loops in MCM5 of both MCM-DH and MCM-SH, the H2I and β-turn loops in MCM7 of MCM-SH, and the β-turn loop in MCM6 of MCM-SH are partially or mostly disordered.

Extended Data Fig. 6 Nucleotide-binding states within human MCM2-7 before and after loading onto DNA.

a) Comparison of nucleotide occupancies at the AAA+ interfaces in the loaded MCM single and double hexamers and the open-gate MCM2-7 dimer (pre-loading state) observed in this study with those in the previously published human MCM double hexamer structure (PDB 7W1Y26). ATPase sites are viewed from the N-terminal MCM tier. b to d) Zoomed views of the ATP binding sites in the loaded MCM double hexamer (in b), the loaded MCM2-7 single hexamer (in c), and open-gate MCM2-7 dimer in the pre-loading state (in d). ATP and magnesium are shown in stick representation and as spheres, respectively, with corresponding cryo-EM map density as grey mesh. Note that the nucleotide occupancy at the MCM2/5 site in the open-gate MCM2-7 (in d) could not be determined (n.d.) due to poor map quality in this region.

Extended Data Fig. 7 Open-ring human MCM2-7 hexamers can dimerize without loading onto DNA.

a) Size exclusion chromatography of purified human MCM2-7 shows predominant dimerization of MCM2-7 hexamers (untagged). Fusing MBP to the N-terminus of MCM5 prevents dimer formation. The elution of molecular weight markers (thyroglobulin – 670 kDa, γ-globulin – 158 kDa) is indicated by arrowheads. b) Negative-stain EM 2D class averages of MCM2-7 dimers, which are structurally distinct from loaded MCM double hexamers (class average shown for comparison). c to h) Cryo-EM data processing and validation of human MCM2-7 dimers. c) Cryo-EM 2D class averages of MCM2-7 dimers without crosslinking. Fuzzy density (outlined by dotted line) indicates conformational flexibility between both hexamers in the MCM dimer. d) Cryo-EM image and e) 2D cryo-EM class averages of crosslinked (with glutaraldehyde) MCM dimers. f) Cryo-EM data processing workflow for 3D reconstruction. g and h) Angular distribution plots for the MCM2-7 dimer (in g, C1 refined) and the locally refined, symmetry expanded MCM2-7 hexamer (in h). We note that although MCM2-7 was mixed with CDT1 prior to cryo-EM sample preparation, no density is observed for this licensing factor, consistent with prior findings that the human proteins, unlike the S. cerevisiae counterparts8,82, do not stably co-associate into an MCM2-7•Cdt1 heptamer83.

Extended Data Fig. 8 Resolution estimation of MCM2-7 cryo-EM reconstructions in the pre-loading state and structural comparison with the loaded MCM double hexamer.

a) Surface view of sharpened cryo-EM map of the full MCM dimer. b) 3D FSC plot for cryo-EM reconstruction of the full MCM dimer. c) Surface view of sharpened cryo-EM map of locally refined MCM hexamer in the MCM dimer. d) 3D FSC plot for cryo-EM reconstruction of locally refined MCM dimer map. e) FSC curves comparing cryo-EM maps and refined models. f to i) The MCM dimer is stabilized by similar interactions between MCM3, MCM5, and MCM7 as in the loaded MCM double hexamer. f and g) MCM5/3/7 dimerization interface in the open-gate MCM dimer. A structure overview is shown in f and zoomed views of the contact sites between subunits in the two hexamers (i-iv) in g. h and i) MCM5/3/7 dimerization interface in the loaded MCM double hexamer. A structure overview is shown in h and zoomed views of the contact sites between subunits in the two hexamers (i-iv) in i. MCM subunits are shown as cartoon, with transparent cryo-EM map density overlaid in one of the hexamers.

Extended Data Fig. 9 Human MCM2-7 can dimerize on DNA in the absence of other loading factors.

a) MCM2-7 dimers with and without nucleotide or CDT1. Incubations were done on ice. b) MCM2-7 dimers and monomers are in a temperature-regulated equilibrium, with a subset of MCM2-7 remaining in the dimeric form (arrowheads) at 37 °C. c) MCM2-7 with an MBP-TEV tag at the N-terminus of MCM5 is monomeric (on ice). Subregions of negative-stain electron micrographs are shown in a–c. d) The MCM5 N-terminus of one hexamer is buried in the adjacent hexamer in both the MCM dimer and loaded MCM-DH. e–h) MBP-MCM5 supports loading of MCM-SHs but not MCM-DH formation. e) Experimental workflow. f) Silver-stained SDS-PAGE gels of inputs and elutions from recruitment and loading reactions with untagged (UT) or MBP-MCM5 MCM2-7 assemblies, either with or without pre-incubation of MCM2-7 with TEV. g) Subregions of negative-stain electron micrographs of elutions from reactions in f. MCM-SH and MCM-DH are marked by magenta and blue arrowheads. h) ORC6 is not required for MCM-SH loading. Left: Experimental setup. Right: Silver-stained SDS-PAGE gels of reactions with MBP-MCM2-7 without or with ORC6. i) Workflow for experiments in j–l. j) Quantification of MCM-DH particles eluted after 0.5 M or 1 M KCl washes. MBP-MCM was pre-treated with TEV before the loading reaction. k) Quantification of MCM-DH particles eluted after 0.5 M or 1 M KCl washes. MCM-SH were first loaded and dimerization initiated by TEV cleavage after removal of ORC, CDC6, CDT1, and free MCM. l) Quantification of MCM-DH particles formed by dimerization of independently loaded MCM-SH, loaded either with full-length (FL) or truncated (Δ) ORC and CDT1. n = 10 electron micrographs for each of three independent experiments in j–l (total n = 30). Black lines represent means in j–l. m–n) Subregion of electron micrographs from k and l showing MCM-DHs (white arrows). For gel source data, see Supplementary Information.

Source data

Extended Data Fig. 10 Cryo-EM data processing and validation of human MCM2-7 formed by dimerization of independently loaded MCM single hexamers in the absence of other loading factors.

a) Representative cryo-EM image. b) 2D cryo-class averages of MCM-DH. c) Workflow of cryo-EM data processing. d) Angular distribution plot for reconstructed cryo-EM volume. e) 3D FSC plot for MCM-DH reconstruction. f) Surface and cut-through views of unsharpened cryo-EM map of MCM-DH formed by dimerization of independently loaded MCM-SHs. g) FSC curve comparing cryo-EM map and refined MCM-DH model.

Extended Data Table 1 Cryo-EM data collection, refinement and validation statistics Full size table Supplementary information Supplementary Information

Uncropped gel images and example electron micrographs.

Reporting Summary Source data Source Data Fig. 2 Source Data Fig. 3 Source Data Fig. 4 Source Data Fig. 5 Source Data Fig. 6 Source Data Extended Data Fig. 1 Source Data Extended Data Fig. 2 Source Data Extended Data Fig. 3 Source Data Extended Data Fig. 9 Rights and permissions

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Yang, R., Hunker, O., Wise, M. et al. Multiple mechanisms for licensing human replication origins. Nature 636, 488–498 (2024). https://doi.org/10.1038/s41586-024-08237-8

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Received 15 February 2024

Accepted 16 October 2024

Published 27 November 2024

Issue Date 12 December 2024

DOI https://doi.org/10.1038/s41586-024-08237-8

Subjects Cryoelectron microscopy DNA-binding proteins DNA replication This article is cited by Mechanisms for licensing origins of DNA replication in eukaryotic cells Bruce StillmanJohn F. X. DiffleyJanet H. Iwasa

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