Abstract
In preparation for bidirectional DNA replication, the origin recognition complex (ORC) loads two hexameric MCM helicases to form a head-to-head double hexamer around DNA1,2. The mechanism of MCM double-hexamer formation is debated. Single-molecule experiments have suggested a sequential mechanism, in which the ORC-dependent loading of the first hexamer drives the recruitment of the second hexamer3. By contrast, biochemical data have shown that two rings are loaded independently via the same ORC-mediated mechanism, at two inverted DNA sites4,5. Here we visualize MCM loading using time-resolved electron microscopy, and identify intermediates in the formation of the double hexamer. We confirm that both hexamers are recruited via the same interaction that occurs between ORC and the C-terminal domains of the MCM helicases. Moreover, we identify the mechanism of coupled MCM loading. The loading of the first MCM hexamer around DNA creates a distinct interaction site, which promotes the engagement of ORC at the N-terminal homodimerization interface of MCM. In this configuration, ORC is poised to direct the recruitment of the second hexamer in an inverted orientation, which is suitable for the formation of the double hexamer. Our results therefore reconcile the two apparently contrasting models derived from single-molecule experiments and biochemical data.
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Acknowledgements
We thank past and present members of the Costa laboratory for useful discussions; G. Coster, A. McClure, C. Kurat, A. Early and L. Drury for sharing reagents and purification protocols; S. Webb and N. Turner for help with biochemical and electron microscopy experiments; A. Nans for support on the Titan Krios; R. Carzaniga (Electron Microscopy STP) for support on the Tecnai G2 Spirit electron microscope; P. Rosenthal for advice; A. Purkiss and P. Walker (Structural Biology STP) for computational support; and N. Patel, A. Alidoust and D. Patel (Fermenation STP) for yeast cultures. This work was funded jointly by the Wellcome Trust, MRC and CRUK at the Francis Crick Institute (FC001065 and FC001066). A.C. receives funding from the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation programme (grant agreement no. 820102). This work was also funded by a Wellcome Trust Senior Investigator Award (106252/Z/14/Z) and a European Research Council Advanced Grant (669424-CHROMOREP) to J.F.X.D.
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Contributions
T.C.R.M. and A.C. conceived the study. T.C.R.M. designed biochemistry experiments. T.C.R.M., J.F.G. and J.L. prepared biochemical reagents and developed the assays. T.C.R.M., J.F.G. and J.L. performed negative-stain imaging. T.C.R.M. and J.L. performed cryo-EM imaging. T.C.R.M. performed all image processing and atomic model building and developed the ReconSil method. J.F.X.D. provided reagents. A.C. supervised the study. T.C.R.M. and A.C. wrote the manuscript with input from J.F.X.D. and the other authors.
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Extended data figures and tables
Extended Data Fig. 1 Nucleosomes function as roadblocks that limit the linear diffusion of double hexamers.
a, Loaded MCM particles are retained on short naked DNA when washed with low salt (300 mM NaOAc, LSW), whereas they slide off DNA when washed with high salt (500 mM NaCl, HSW). When chromatinized, the same DNA substrate retains MCM particles. In this experiment, soluble MCM loading reactions are bound to streptavidin-coated magnetic beads via a desthiobiotin tag on the origin DNA, before removing unbound proteins with high or low salt washes. b, Electron microscopy imaging of soluble MCM loading reactions yields 2D class averages of licensing complexes on chromatinized DNA. Despite the sample heterogeneity, recognizable classes can be obtained for ORC, MCM–Cdt1, nucleosomes, ORC that maps in close proximity to a nucleosome, as well as double hexamers. c, Comparison between MCM loading on chromatinized DNA and DNA containing M.HpaII (MH) roadblocks. After high-salt-wash treatment, equal amounts of loaded MCM helicases are retained on streptavidin beads, which indicates that nucleosomes are not required for efficient formation of double hexamers in this assay. d, Yeast replication origins centred on ARS1, containing the two inverted ORC binding sites, ACS (high affinity, red arrow) and B2 (low affinity, orange arrow). ARS1 is flanked by nucleosomes, covalently attached methyltransferases (M.HpaII-M.HpaII) or a combination of the two to obtain asymmetric origins with recognizable features that mark the ends of the origin (M.HpaII–nucleosome and nucleosome–M.HpaII). For gel source data for a and c, see Supplementary Fig. 1.
Extended Data Fig. 2 In silico reconstitution of origin licensing performed on asymmetric origins of replication.
a, Cartoon depicting the ReconSil procedure, as performed to investigate the interactions between ORC and an asymmetric origin. Particles are picked on micrographs with a low signal-to-noise ratio. Two-dimensional averages are calculated. Averages are superposed to the raw micrographs, overlaid to the particles that contributed to their generation. For this purpose, particle coordinates are combined with alignment parameters derived from 2D classification. This approach yields a signal-enhanced view of single instances of molecular complexes bound to a flexible substrate (in this case, ORC binding to an entire origin of replication). b, Representative raw micrograph, 2D class averages positioned according to their constituent particles, and a micrograph of origins reconstituted in silico with positioned 2D class averages overlaid onto the original image. Instances boxed in black are selected, red are rejected. c, Left, origins might be rejected owing to local particle clustering and aggregation, or because they contain visible raw particles that could not be classified (and therefore are not matched by a high-quality 2D average). This assay used M.HpaII–nucleosome origins that permit measurement of the length of origins because both the M.HpaII roadblock (next to ACS-bound ORC) and the nucleosome can be reconstituted. The measurement of origins reconstituted in silico was performed using ImageJ. d, Comparison of raw negative-stain electron microscopy data and origins reconstituted in silico for representative OCCM-bound origins shown in Fig. 1g. e, Example of origins reconstituted in silico (and corresponding raw images) showing double hexamers recruited to nucleosome–M.HpaII origins. ORC frequently rebinds to the ACS on origins that contain double hexamers, but shows no fixed interaction with the C-terminal face of the loaded double hexamer.
Extended Data Fig. 3 Cryo-EM structure of the MO loading intermediate.
a, The MO intermediate is enriched when MCM–Cdt1 concentration is limiting, as quantified using negative-stain electron microscopy. An MCM loading reaction performed for 30 min in the presence of excess MCM–Cdt1 results in the majority of MCM helicases forming double hexamers on DNA. If MCM–Cdt1 concentration is limited and loading time is reduced (7 min), MO complexes form but do not mature into double hexamers; this indicates that the MO intermediate is on the path to the formation of the double hexamer. Bar chart shows mean, n = 2 independent experiments. b, Example of an aligned movie. c, Resulting 2D averages. d, Angular distribution. e, Resolution estimated using gold-standard Fourier shell correlation. f, Three rotated views and a cut-through view of the MO 3D structure, colour-coded according to local resolution. g, Structure obtained by multi-body refinement, displayed as described for e.
Extended Data Fig. 4 Pipeline for generating the MO structure.
Schematic shows the classification and refinement of the MO cryo-EM map.
Extended Data Fig. 5 A role for Orc6 in modulating MCM loading.
a, Two elements connect ORC and the N-terminal face of MCM. One is Orc6 and the second is DNA, which is solvent-exposed between the ORC and MCM complexes owing to the bend induced by complex formation. b, Orc6 contains a domain architecture preserved in the related TFiiB transcription factor29. Although the precise mode of DNA engagement for the N- and C-terminal domains of TFiiB and Orc6 differ, notable conservation can be detected. c, Sequence alignment between the N-terminal domain of TFiiB and the N-terminal domain of Orc6. The N-terminal domain of Orc6 contacts DNA through a conserved lysine that is also found in TFiiB. Mutation of the equivalent lysine in Drosophila Orc6 affects DNA binding in vitro, as well as replication in extracts and cells30. d, A conserved helix18,31 of the Orc6 C-terminal domain (Orc6C) touches the N-terminal helical bundle of Mcm5. The Orc6 N-terminal domain (Orc6N) touches the N-terminal helical bundle of Mcm2. Together, the N-terminal and C-terminal domains of Orc6 latch across the Mcm2–Mcm5 gate. e, No steric clash can be detected between Orc6 and Cdt1 when MO and MCM–Cdt1 are superposed via the N-terminal domain of Mcm2. However, the C-terminal domain of Orc6 severely clashes with the N-terminal domain of Mcm5 in this configuration. Only Orc6 from MO is shown in the MCM–Cdt1 superposed structure.
Extended Data Fig. 6 Structure of ORC–DNA in different states.
a–c, Comparison between the cross-linked ORC–DNA complex imaged in isolation (a), ORC–DNA in the OCCM complex (b) and ORC–DNA in the MO complex (c). Nucleotide occupancy appears the same in all three cases. It should be noted, however, that ORC–DNA alone and within the OCCM complex were co-incubated with ATPγS, whereas ORC in MO was imaged in ATP. Orc2 in ORC–DNA contains a visible winged-helix domain (WHD) that topologically closes ORC around DNA. ORC in OCCM is Cdc6-engaged. The Orc2 winged-helix domain is virtually absent in the cryo-EM map of the MO, which indicates that this domain is flexible. This discrepancy might reflect a different ORC configuration in MO, or the fact that the previously published ORC–DNA structure was stabilized by glutaraldehyde crosslinking. Despite Cdc6 being present in the sample, ORC in MO is not Cdc6-bound.
Extended Data Fig. 7 OC–MC contains a recruited, but not DNA-engaged, MCM–Cdt1.
a, b, Cryo-EM 2D class averages indicate that OC–MC is a pre-OCCM intermediate. c, d, This finding is confirmed by comparison of raw origins and origins reconstituted in silico, which permit visualization of the DNA path through OC–MC (c) and OCCM (d) in negative-stain experiments. In OC–MC, MCM–Cdt1 has engaged a DNA-bound ORC complex; however, DNA remains outside the MCM channel. In this configuration, DNA is aligned to the Mcm2–Mcm5 gate, which can be located in the 2D images because of its proximity to the prominent N-terminal lobe of Cdt1 (white arrow). By contrast, DNA runs through the central channels of both ORC and MCM in the OCCM complex, in preparation for Cdt1 release and closure of the MCM ring.
Extended Data Fig. 8 ORC in MO is perfectly positioned for loading the second MCM ring in the correct orientation for the formation of the double hexamer.
a, Negative-stain and cryo-EM 2D classes, and 3D structures of OCCM (top) and MO (bottom), with the loading intermediates aligned via their respective ORC complexes. b, Three-dimensional model, based on a, of the proposed mechanism for recruitment of the second MCM. OCCM is shown superposed to the ORC of MO. This superposition places a second MCM–Cdt1 such that its Mcm2–Mcm5 gate is oriented for threading duplex DNA into the MCM channel. c, Negative-stain 2D class showing a post-MO loading intermediate, captured by supplementing MO complexes with MCM–Cdt1 before imaging. This class appears to be a second MCM recruitment complex, containing MO and an additional MCM–Cdt1. d, A cryo-EM 2D class average of the post-MO complex (top) shows bent duplex DNA aligned to the Mcm2–Mcm5 DNA gate of the second MCM–Cdt1, captured before DNA threading. This is the same configuration that was previously identified for the OC–MC complex (middle). Alignment of the OC–MC and MO 2D classes by their respective ORC complexes matches the observed configuration of the second MCM recruitment complex, MOC–MC. e, Three-dimensional model of MOC–MC, based on the MCM-Cdt1 structure10, the MO structure (this study) and 2D class averages shown in c and d. f, Cdc6 is required for the loading of the second MCM helicase. Following immunodepletion of Flag-tagged Cdc6, MO is unable to load a second MCM; this results in a failure to form salt-stable double hexamers on DNA in the absence of additional Cdc6. For gel source data, see Supplementary Fig. 1.
Extended Data Fig. 9 A second ORC can bind to a loaded MCM helicase before release of the first ORC.
a, Negative-stain 2D class, nucleosome–M.HpaII origin reconstituted in silico, cryo-EM 2D class and schematic showing an intermediate on the path to the formation of the double hexamer. The intermediate contains a single-loaded MCM helicase (Cdt1 has been released) flanked by ORC at its C and N termini (ORC–MCM–ORC, or O2M). The in silico reconstitution shows an entire origin, which spans a nucleosome, an ORC at the C-terminal face of an MCM hexamer, an ORC at the N-terminal face of the MCM and a covalently linked M.HpaII. b, In silico reconstitution and schematic showing a Nucleosome–M.HpaII origin bound by MO. In this configuration the MCM in MO occupies the ACS site, which must have previously been bound by an ORC (as seen in the ORC–MCM–ORC complex in a). This observation demonstrates that MCM sliding towards the nucleosome has occurred.
Supplementary information
Supplementary Information
This file contains discussion of the potential implications of the mechanism described in this study, for MCM helicase loading in higher eukaryotes, and the entire Methods section for the manuscript.
Supplementary Figure
Uncropped blots used in the main figures and extended data figures.
Supplementary Video 1 |Atomic model for the MO complex
Docking of ORC and MCM into the cryo-EM map reveals that Orc6 bridges ORC and the N-terminal domain of MCM. While the structure of the Orc6 C-terminal domain was known, we refined an atomic model for the N-terminal domain, based on the TFiiB homology. We find that the N-Orc6 engages duplex DNA using a conserved Lys also contained in TFiiB.
Supplementary Video 2 | Comparison between the MCM rings in OCCM and the MO intermediate
In OCCM, the MCM ring is Cdt1-bound and the Mcm2-5 DNA gate is open, while in MO the gate is closed. The MCM ring can be extracted from MO and docked into the cryo-EM density for the DNA-bound DH, resulting in an excellent fit. This observation is coherent with the observation that MO and DH contain the same nucleotide state in the MCM ring. We conclude that MO contains a bona fide single loaded MCM ring.
Supplementary Video 3 | Comparison between MO containing wild type ORC or ORC featuring an Orc6 N-terminal truncation (Δ119)
Altering the MCM-ORC interaction interface changes the ORC orientation relative to MCM. The pivot point of this structural rearrangement appears to be Orc6, whose N-terminal domain is highlighted in red in the atomic model.
Supplementary Video 4 | Mechanism of head-to-head DH formation, visualized using cryo-EM 2D averages or atomic models
ORC first binds the ACS site (next to a nucleosome in our construct), it then recruits MCM forming the OC-MC complex, where ORC touches the C-terminal side of the MCM ring. DNA bent by ORC is threaded through the Mcm2-5 gate to form the OCCM. After OCCM disassembly, a new ORC binding event occurs at the N-terminal side of MCM, forming the MO intermediate. Here, ORC engages DNA in an inverted configuration and interacts with the dimerization interface of MCM. In this state, ORC is perfectly poised for the recruitment of a second MCM hexamer via the same elements used to recruit the first MCM ring, forming the MOC-MC intermediate. Finally, the DNA is threaded into the second MCM helicase and the DH forms.
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Miller, T.C.R., Locke, J., Greiwe, J.F. et al. Mechanism of head-to-head MCM double-hexamer formation revealed by cryo-EM. Nature 575, 704–710 (2019). https://doi.org/10.1038/s41586-019-1768-0
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DOI: https://doi.org/10.1038/s41586-019-1768-0
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