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Cloning, expression and purification

ORC, Cdc6, Mcm27Cdt1, DDK, CDK, Sld2, Sld3Sld7, Cdc45, Dpb11, Pol , Pol exo-, Pol , TopoI, Mcm10 and yeast histone octamer were purified on the basis of previously established protocols1,11,24,33,48,49,50,51.

Designed DNA fragments (Supplementary Table 1) were subcloned from pMA vectors (Supplementary Table 2) to pRS shuttle vectors (Supplementary Table 2), which were used to generate yeast strains (Supplementary Table 3) used to overexpress Mcm27Cdt1 mutants. The oMG25 DNA fragment was subcloned from pMG39 to pAM38 using MluI and XbaI restriction sites to obtain pMG69, which was integrated into the yJF21 yeast strain, thus generating the yAE164 strain that was used to overexpress the Mcm2 6A mutant (Mcm2 V580A/K582A/P584A/K587A/W589A/K633A). The oMG27 DNA fragment was subcloned from pMG43 to pJF4 using BsiWI and SphI restriction sites to obtain pMG53, followed by the integration of pMG53 into the yAM20 strain, yielding the yAE160 strain, which was used for overexpression of the Mcm6 2E mutant (Mcm6 T423E/R424E). The oMG28 DNA fragment was subcloned from plasmid pMG44 to pJF4 using BsiWI and SphI restriction sites, thus obtaining plasmid pMG54. The pMG54 plasmid was integrated into the yAM20 strain, yielding the yAE161 strain that was used to overexpress the Mcm6 5E mutant (Mcm6 T408E/Q409E/L410E/G411E/L412E). All Mcm27Cdt1 mutants were purified essentially as wild type50.

A gene block encoding a twin-strep tag and the first three codons of Psf3 was amplified and cloned into pFJD5 by restriction-free cloning techniques. A list of primers and gene blocks used is included in Supplementary Table 1. BL21(DE3)-CodonPlus-RIL cells (Agilent) were transformed with GINS expression plasmid (pJL003). Transformant colonies were inoculated into a 250-ml LB culture containing kanamycin (50gml1) and chloramphenicol 35gml1), which was grown overnight at 37C with shaking at 200rpm. The following morning, the culture was diluted 100-fold into 6 1l of LB with kanamycin (100gml1) and chloramphenicol (35gml1). The cultures were left to grow at 37C until an optical density at 600nm (OD600nm) of 0.5 was reached; 0.5mM isopropyl -d-1-thiogalactopyranoside (IPTG) was added to induce expression and cells were left shaking for 3h. Cells were collected by centrifugation at 4,000rpm for 20min in a JS.4.2 rotor (Beckman). For lysis, cell pellets were resuspended in 120ml of lysis buffer (100mM Tris-HCl pH 8.0, 10% glycerol, 0.02% NP-40, 1mM EDTA, 200mM NaCl, Roche protease inhibitor tablets and 1mM dithiothreitol (DTT) + 0.7mM phenylmethylsulfonyl fluoride(PMSF). The lysate was sonicated for 120s (5s on, 5s off) at 40% on a Sonics Vibra-Cell sonicator. Insoluble material was removed by centrifugation at 20,000rpm for 30min in a JS.25.50 rotor (Beckman). The supernatant was loaded by gravity onto a 1-ml Strep-TactinXT column (IBA). The resin was washed extensively with wash buffer (100mM Tris-HCl pH 8.0, 10% glycerol, 1mM DTT and 1mM EDTA). GINS was eluted by the addition of 6ml of 1 buffer BXT (IBA) supplemented with 10% glycerol and 1mM DTT. The GINS-containing fractions were pooled and dialysed overnight in gel filtration buffer (25mM HEPES-KOH pH 7.6, 10% glycerol, 0.02% NP-40, 200mM potassium acetate and 1mM DTT). The sample was concentrated and loaded onto a HiLoad 16/600 Superdex 200 equilibrated in the same buffer. GINS-containing fractions were pooled, aliquoted and snap-frozen in liquid N2. About 22mg GINS was purified from a 6-litre culture.

The codon-optimized expression sequence for MH containing a HRV 3C protease cleavage site followed by a twin-strep tag was synthesized and cloned into pET302 by GeneWiz Synthesis (pJL004). T7 express cells (NEB) were transformed with pJL004. Transformant colonies were inoculated into a 250-ml LB culture with ampicillin (100gml1), which was grown overnight at 37C with shaking at 200rpm. The following morning, the culture was diluted 100-fold into 6 1l of LB with ampicillin (100gml1). The cultures were left to grow at 37C until an OD600nm of 0.5 was reached; 0.5mM IPTG was added to induce expression and cells were left shaking for 3h. Cells were collected by centrifugation at 4,000rpm for 20min in a JS.4.2 rotor (Beckman). For lysis, cell pellets were resuspended in 80ml of lysis buffer (20mM Tris-HCl pH 8.5, 10% glycerol 0.5mM EDTA, 500mM KCl, Roche protease inhibitor tablets and 2mM tris(2-carboxyethyl)phosphine (TCEP)) + 0.7mM PMSF. The lysate was sonicated for 120s (5s on, 5s off) at 40% on a Sonics Vibra-Cell sonicator. Insoluble material was removed by centrifugation at 20,000rpm for 30min in a JS.25.50 rotor (Beckman). The supernatant was loaded by gravity onto a 5-ml Strep-TactinXT column (IBA). The resin was washed extensively with lysis buffer. MH was eluted by the addition of 12ml of 1 BXT (IBA) supplemented with 10% glycerol and 1mM DTT. The MH-containing fractions were pooled and loaded onto a HiLoad 16/600 Superdex 75 equilibrated in gel filtration buffer (20mM Tris-HCl pH 8.5, 10% glycerol 0.5mM EDTA, 100mM KCl and 0.5mM TCEP). MH-containing fractions were pooled, aliquoted and snap-frozen in liquid N2. About 36mg MH was purified from a 6-litre culture.

The native ARS1 origin of replication flanked by Widom 601 and 603 sites or MH-flanked was amplified by PCR and purified as previously described24. The 6 ARS1 array (pSSH005) was assembled by inserting an array of 6 ARS1 origins with 40-bp spacing flanked by MH sites using NEBuilder HiFi assembly. The 6 ARS1 origin array was amplified from pSSH005 using primer oSSH038 and concentrated by ethanol precipitation. A list of primers and DNAs used is included in Supplementary Table 1.

Soluble yeast nucleosomes were reconstituted from octamers and DNA by salt gradient dialysis in several steps from 2 to 0.2 M NaCl as previously described24. Following nucleosome refolding, a final dialysis step was performed into loading buffer (25mM HEPES-KOH pH 7.6, 80mM KCl, 100mM sodium acetate, 0.5mM TCEP) and loaded onto a Superose 6 Increase 3.2/300 column equilibrated in the same buffer. Fractions containing ARS1 origin DNA bound by 2 nucleosomes were pooled, concentrated, and stored at 4C. Reconstitution conditions were optimized by small-scale titration and nucleosomes checked by 6% native PAGE.

The conjugation of MH with origin substrates was performed in 50mM Tris-HCl pH 8.0, 1mM EDTA and 0.5mM 2-mercaptoethanol supplemented with 100M S-adenosylmethionine (NEB). The reaction was carried out overnight at 30C, with a 10:1 molar ratio of MH:DNA. After conjugation, reactions were centrifuged at 14,680rpm for 5min and loaded onto a 1ml RESOURCE-Q column equilibrated into DNA buffer (50mM Tris-HCl pH 8.0 and 5mM 2-mercaptoethanol). MH-conjugated DNA was eluted in a linear gradient of DNA buffer B (50mM Tris-HCl pH 8.0, 5mM 2-mercaptoethanol and 2M NaCl) over 24column volumes. Fractions containing MH-conjugated DNA were pooled, concentrated and stored at 80C. Conjugations were checked by 6% native PAGE.

The conjugation of MH with origin substrates was performed in 25mM Tris-HCl pH 7.5, 10mM magnesium acetate, 50mM potassium acetate and 1mgml1 BSA supplemented with 150M S-adenosylmethionine (NEB). The reaction was carried out at 32C for 1h then overnight at 4C, with a 20:1 molar ratio of MH:DNA. After conjugation, reactions were centrifuged at 14,680rpm for 5min and loaded onto a Superose 6 Increase 10/300 column equilibrated into array buffer (25mM HEPES-KOH pH 7.5, 200mM NaCl and 1mM DTT). Fractions containing MH-conjugated array DNA were pooled, concentrated and stored at 4C. Conjugations were checked by 6% native PAGE.

The 616-bp ARS1 circles were assembled and prepared as previously described1 with the following modifications. The dephosphorylation step was performed with the use of quickCIP, instead of Antarctic phosphatase, for 30min at 37C followed by enzyme inactivation at 80C for 2min. After the ligation step, the DNA was concentrated as described and incubated with T5 exonuclease (NEB; 37C for 1h) to eliminate non-ligated DNA. Ethanol precipitation, agarose electrophoresis and electroelution were omitted; instead, phenol/chloroform/isoamyl-alcohol extraction was performed, followed by ethanol precipitation using sodium acetate (pH 5.1) and the neutral carrier GeneElute Linear Polymer (LPA, MERCK).

ARS1 nucleosome-flanked origin DNA (20nM) was incubated with 52nM ORC, 52nM Cdc6 and 110nM Mcm27Cdt1 for 30min at 24C in loading buffer (25mM HEPES-KOH pH 7.6, 100mM potassium glutamate, 10mM magnesium acetate, 0.02% NP-40 and 0.5mM TCEP) + 5mM ATP. The reaction was supplemented with 80nM DDK, and incubation continued for a further 10min at 24C. Nucleoprotein complexes were isolated by incubation with 5l MagStrep type3 XT beads (IBA) pre-washed in 1 loading buffer for 30min at 24C. The beads were washed three times with 100l wash buffer (25mM HEPES-KOH pH 7.6, 105mM potassium glutamate, 5mM magnesium acetate, 0.02% NP-40 and 500mM NaCl) and once with 100l loading buffer. Loaded, phosphorylated double hexamers were eluted in 20l elution buffer (25mM HEPES-KOH pH 7.6, 105mM potassium glutamate, 10mM magnesium acetate, 0.02% NP-40, 0.5mM TCEP, 27mM biotin and 5mM ATP) for 10min at 24C. The remaining supernatant was removed and incubated with 200nM CDK for 5min at 30C. A mix of firing factors was then added to a final concentration of 30nM Dpb11, 100nM GINS, 80nM Cdc45, 20nM Pol , 30nM Sld3Sld7 and 50nM Sld2. After 30min of incubation, the reaction was applied directly to grids or diluted fivefold in 1 loading buffer for ReconSil experiments.

MH-capped ARS1 array DNA (5nM) was incubated with 52nM ORC, 52nM Cdc6 and 110nM Mcm27Cdt1 for 30min at 24C in loading buffer (25mM HEPES-KOH pH 7.6, 100mM potassium glutamate, 10mM magnesium acetate, 0.02% NP-40 and 0.5mM TCEP) + 5mM ATP. The reaction was supplemented with 80nM DDK, and incubation continued for a further 10min at 24C. Nucleoprotein complexes were isolated by incubation with 5l MagStrep type3 XT beads (IBA) pre-washed in 1 loading buffer for 30min at 24C. The beads were washed three times with 100l wash buffer (25mM HEPES-KOH pH 7.6, 105mM potassium glutamate, 5mM magnesium acetate, 0.02% NP-40 and 500mM NaCl) and once with 100l loading buffer. Loaded, phosphorylated double hexamers were eluted in 20l elution buffer (25mM HEPES-KOH pH 7.6, 105mM potassium glutamate, 10mM magnesium acetate, 0.02% NP-40, 0.5mM TCEP, 27mM biotin and 5mM ATP) for 10min at 24C. The remaining supernatant was removed and incubated with 200nM CDK for 5min at 30C. A mix of firing factors was then added to a final concentration of 90nM Dpb11, 300nM GINS, 240nM Cdc45, 60nM Pol , 90nM Sld3Sld7 and 150nM Sld2. After 30min of incubation, the reaction was diluted fivefold in 1 loading buffer and applied to grids.

For experiments in which DNA was partially digested after the CMG formation reaction, MseI (NEB) was added at a concentration of 0.1U diluted in 1 loading buffer. Incubation was performed for 10min at 30C before applying to EM grids.

Replication assays were performed as described previously52. The reactions were incubated in a ThermoMixer at 30C with 1,250rpm shaking. The reaction buffer was as follows: 25mM HEPES-KOH pH 7.6, 10mM magnesium acetate, 2mM DTT, 0.02% NP-40, 100mM potassium glutamate and 5mM ATP. MCM helicase loading reaction (5l) contained 30nM ORC, 30nM Cdc6, 60nM Mcm27Cdt1 (or MCM mutants) and either 4nM ARS-containing 10.6kb supercoiled plasmid (pJY22; Supplementary Table 2) or 40nM ARS-containing short linear DNA (flanked by nucleosomes or MH; Supplementary Table 2) as for Fig. 1. After 20min, DDK was added to a final concentration of 50nM and further incubated for 20min. Next, the reaction volume was doubled (final volume was 10l) by adding proteins (20nM Pol , 30nM Dpb11, 20nM GINS, 50nM Cdc45, 20nM CDK, 50nM RPA, 10nM TopoI, 100nM Pol , 25nM Sld3Sld7, 10nM Mcm10 and 50nM Sld2) and nucleotides (200M CTP, 200M GTP, 200M UTP, 80M dCTP, 80M dGTP, 80M dTTP, 80M dATP and 50nM 32P-dCTP). For replication reactions with linear DNA (Fig. 1) Pol exo- was used instead of Pol wild type to reduce end labelling and the concentration of deoxynucleotides was modified (that is, 30M dCTP, 30M dGTP, 30M dTTP, 30M dATP and 100nM 32P-dCTP). The reactions were stopped by EDTA after 15 and 30min for reactions with 10.6-kb supercoiled DNA or after 20min for reactions with short linear DNA substrates and processed as described51,52. The replication products were separated using 0.8% agarose alkaline gel for 17h at 25V for reactions with 10.6-kb supercoiled DNA. For reactions with short DNA substrates, samples were separated using 2% agarose alkaline gel for 4h at 38V. The image signal from Fig. 1e was background-subtracted in Fiji using the subtract background algorithm in Fiji v.2.0.0 (ref. 53).

The experiment was performed as described previously1. The concentrations of proteins were as follows: 10nM ORC, 50nM Cdc6, 100nM Mcm27Cdt1 (or Mcm mutants), 80nM DDK for the helicase loading step (5l) and 20nM Pol , 30nM Dpb11, 40nM GINS, 50nM Cdc45, 30nM CDK, 10nM TopoI, 25nM Sld37, 5nM Mcm10, 50nM Sld2 for the helicase activation step (10l). Radiolabelled 616-bp circular DNA (25fmol) was used. After processing the reactions as described previously1, Ficoll 400 (final concentration was 2.5%) and Orange G were used to load the sample onto a native 3.5% bis-polyacrylamide gel (1 TBE) and separation was carried out for 21h at 90V using Protean II XL Cell apparatus (Bio-Rad) at room temperature. The 0.7-mm gel was dried (without fixation) at 80C for 105min, exposed to a phosphor screen and scanned with the use of Typhoon phosphor imager.

NS-EM sample preparation was performed on 400-mesh copper grids with carbon film (Agar Scientific). Grids were glow-discharged for 30s at 45mA using a K100X glow discharge unit (Electron Microscopy Sciences) before a 4-l sample was applied to the grids and incubated for 2min. Grids were stained by two successive applications of 4l 2% (w/v) uranyl acetate with blotting between the first and second application. Stained grids were blotted after 20s to remove excess stain. Unless described otherwise, data collection was carried out on a Tecnai LaB6 G2 Spirit transmission electron microscope (FEI) operating at 120keV. A 2K2K GATAN Ultrascan 100 camera was used to collect micrographs at a nominal magnification of 30,000 (with a physical pixel size of 3.45 per pixel) within a 0.5 to 2.0m defocus range.

A subset of particles was manually picked using RELION-3.1 (ref. 26) and used as a training dataset for Topaz training53. Subsequent image processing was performed using RELION-3.1. The CTF of each micrograph was estimated using Gctf (ref. 54) and particles were extracted and subjected to reference-free 2D classification in RELION-3.1.

For ReconSil experiments, image processing was carried out as detailed above. Reference-free 2D classification in RELION generates both 2D class averages and star files detailing the class assignment, particle coordinates and transformations (translations and rotations) applied to the raw particles for alignment.2D averages are superposed on the raw micrographs, overlaid on the particles that contributed to their generation. This yieldedsignal-enhanced ReconSiled micrographs reconstituting the contextof complete origins of replication. ReconSiled micrographs were used for the selection and rejection of origin nucleoproteins for further analysis.

ReconSiled origins were analysed as previously described24. In brief, ReconSiled micrographs were used to re-extract particles of interest in RELION. Selected particles were manually classified for statistical analysis. Measurements of ReconSiled origins were performed manually using Fiji55 and plotted in GraphPad Prism v.9.2.0.

CMG assembly reactions (reconstituted as described in In vitro CMG assembly on short chromatinized origins) were frozen on 400-mesh lacey grids with a layer of ultra-thin carbon (Agar Scientific). All grids were freshly glow-discharged for 1min at 45mA using a K100X glow discharge unit (Electron Microscopy Sciences) before plunge freezing. Samples were prepared by applying 4l of undiluted CMG assembly reactions for 2min on a grid equilibrated to 25C in 90% humidity. The grid was blotted for 4.5s and plunged into liquid ethane. Data collection was performed on an in-house Thermo Fisher Scientific Titan Krios transmission electron microscope operated at 300kV, equipped with a Gatan K2 direct electron detector camera (Gatan) and a GIF Quantum energy filter (Gatan). Images were collected automatically using the EPU software (Thermo Fisher Scientific) in counting mode with a physical pixel size of 1.08 per pixel, with a total electron dose of 51.4 electrons per 2 during a total exposure time of 10s dose-fractionated into 32 movie frames (Extended Data Table 1). We used a slit width of 20eV on the energy filter and a defocus range of 2.0 to 4.4m. A total of 65,286 micrographs were collected from two separate sessions.

Data processing was performed using RELION-3.1 (ref. 26) and cryoSPARC v.3.2 (ref. 56) (Extended Data Fig. 3). The movies for each micrograph were first corrected for drift and dose-weighted using MotionCorr2 (ref. 57). CTF parameters were estimated for the drift-corrected micrographs using Gctf within RELION-3.1 (ref. 54). Dataset one was first processed separately and combined with dataset two at a later stage.

For the first dataset, particles were picked using a manually curated particle set as a template in crYOLO v.1.7.5 (ref. 58). These particles were binned by 2 and extracted with a box size of 360 pixels for 2D and 3D classification. A subset of 1,600 representative particles across the entire defocus range was selected. Picks in areas of obvious particle aggregation were removed along with particles located on the carbon lace. A Topaz53 model was then iteratively trained on the remaining particles. All particles were re-picked with the Topaz model with the default score threshold of 0 for particle prediction. The two datasets were combined and a total of 927,109 particles were picked, binned by 2 and extracted with a box size of 360 pixels. We carried out 2D classification to remove remaining smaller particles and contaminants. We subjected the remaining particles to 3D multi-reference classification with four sub-classes, angular sampling of 7.5, a regularization parameter T of 5 using low-pass-filtered initial models from previous ab initio and processing steps on dataset 1 of dCMGE complexes, and double hexamer model generated from EMD-3960 (Extended Data Fig. 3). The resulting 133,262 (trans-dCMGE) and 46,049 (cis-dCMGE) particles with density corresponding to Pol on both CMG molecules were un-binned and refined to yield maps with resolutions of 7.7 and 14.4. C2 symmetry imposition did not improve the quality of the maps. The 133,262 trans-dCMGE particles were imported into cryoSPARC and subjected to multiple rounds of non-uniform refinement, heterogenous 3D classification and non-uniform local refinement, yielding a map at approximately 8 (Extended Data Fig. 3). Attempts to improve cis-dCMGE were unsuccessful given the limited particle numbers. As expected, these reconstructions do not show secondary structural features owing to the conformational heterogeneity between the two CMGE molecules bound by flexible DNA. We applied a C2 symmetry expansion procedure to both trans- and cis-dCMGE particles (179,311) with re-centring on one CMGE in RELION and combined all particles. We also downsized the box size to 512 pixels during this process to speed up downstream processing. Following this, masked 3D refinement with local searches in C1 of the centred single CMGE (consisting of 358,622 particles) was refined to 4.2- resolution. These particles were subjected to several rounds of CTF refinement and two rounds of Bayesian polishing. After this, CTF-refined and polished particles were refined with local searches in C1 with a mask encompassing the entire CMGE density to 3.6- resolution. To better resolve the DNA inside the MCM central channel, densities corresponding to Cdc45, GINS and Pol were subtracted in RELION. Signal-subtracted particles were analysed by 3D variability analysis in cryoSPARC (ref. 56). A subset of 71,348 particles was selected based on the quality of DNA density. These signal-subtracted particles were subsequently reverted to the original particles and refined using local searches in C1 using local searches to 3.5- resolution.

All refinements were performed using fully independent data half-sets and resolutions are reported based on the Fourier shell correlation (FSC)=0.143 criterion (Extended Data Fig. 2). FSCs were calculated with a soft mask. Maps were corrected for the modulation transfer function of the detector and sharpened by applying a negative B-factor as determined by the post-processing function of RELION or in cryoSPARC. The final RELION half-maps were used to produce a density modified map using the PHENIX Resolve CryoEM (refs. 28,59). This 3.4- map showed significant improvements for side chain and DNA density as well as for overall interpretability. Local-resolution estimates were determined using PHENIX or cryoSPARC (Extended Data Fig. 2f,j). The conversions between cryoSPARC and RELION files were performed using the UCSF pyem v.0.5 package60.

CMG (from PDB 6SKL)31, Pol2 subunit (from PDB 6HV9)33 and a homology model of the N-terminal domain of Dpb2 obtained from the Phyre2 server61 were docked initially into the cryo-EM map produced from Resolve CryoEM, using USCF Chimera, and refined against the map using Namdinator62 as a starting point for modelling with Coot v.0.9.1 (ref. 63). The DNA and the MCM5 winged helix domain were built de novo. The register of origin DNA engagement of dCMGE is heterogeneous because MCM double hexamers can slide along duplex DNA before dCMGE is formed. For this reason we could not build the origin DNA sequence with certainty and modelled polyA:polyT DNA instead. The resulting model was then subjected to an iterative process of real-space refinement using Phenix.real_space_refinement64 with geometry and secondary structure restraints and base-pairing and base-stacking restraints where appropriate, followed by manual inspection and adjustments in Coot. The geometries of the atomic model were evaluated by the MolProbity webserver65.

Maps were visualized in UCSF Chimera66 and ChimeraX67 and all model illustrations and morphs were prepared using ChimeraX or PyMOL.

Statistical analysis was performed using a two-tailed Welchs t-test in GraphPad Prism v.9.2.0. No statistical methods were used to predetermine sample size. The experiments were not randomized, and investigators were not blinded to allocation during experiments and outcome assessment.

Further information on research design is available in theNature Research Reporting Summary linked to this paper.

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Mechanism of replication origin melting nucleated by CMG helicase assembly - Nature.com