Cloning, expression and purification
ORC, Cdc6, Mcm2–7–Cdt1, DDK, CDK, Sld2, Sld3–Sld7, Cdc45, Dpb11, Pol ε, Pol ε exo-, Pol α, TopoI, Mcm10 and yeast histone octamer were purified on the basis of previously established protocols1,11,24,33,48,49,50,51.
Cloning, expression and purification of Mcm2–7–Cdt1 mutants
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 Mcm2–7–Cdt1 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 Mcm2–7–Cdt1 mutants were purified essentially as wild type50.
Cloning, expression and purification of GINS
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 (50 µg ml−1) and chloramphenicol 35 µg ml−1), which was grown overnight at 37 °C with shaking at 200 rpm. The following morning, the culture was diluted 100-fold into 6× 1 l of LB with kanamycin (100 µg ml−1) and chloramphenicol (35 µg ml−1). The cultures were left to grow at 37 °C until an optical density at 600 nm (OD600 nm) of 0.5 was reached; 0.5 mM isopropyl β-d-1-thiogalactopyranoside (IPTG) was added to induce expression and cells were left shaking for 3 h. Cells were collected by centrifugation at 4,000 rpm for 20 min in a JS.4.2 rotor (Beckman). For lysis, cell pellets were resuspended in 120 ml of lysis buffer (100 mM Tris-HCl pH 8.0, 10% glycerol, 0.02% NP-40, 1 mM EDTA, 200 mM NaCl, Roche protease inhibitor tablets and 1 mM dithiothreitol (DTT) + 0.7 mM phenylmethylsulfonyl fluoride (PMSF). The lysate was sonicated for 120 s (5 s on, 5 s off) at 40% on a Sonics Vibra-Cell sonicator. Insoluble material was removed by centrifugation at 20,000 rpm for 30 min 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 (100 mM Tris-HCl pH 8.0, 10% glycerol, 1 mM DTT and 1 mM EDTA). GINS was eluted by the addition of 6 ml of 1× buffer BXT (IBA) supplemented with 10% glycerol and 1 mM DTT. The GINS-containing fractions were pooled and dialysed overnight in gel filtration buffer (25 mM HEPES-KOH pH 7.6, 10% glycerol, 0.02% NP-40, 200 mM potassium acetate and 1 mM 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 22 mg GINS was purified from a 6-litre culture.
Cloning, expression and purification of MH
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 (100 µg ml−1), which was grown overnight at 37 °C with shaking at 200 rpm. The following morning, the culture was diluted 100-fold into 6× 1 l of LB with ampicillin (100 µg ml−1). The cultures were left to grow at 37 °C until an OD600 nm of 0.5 was reached; 0.5 mM IPTG was added to induce expression and cells were left shaking for 3 h. Cells were collected by centrifugation at 4,000 rpm for 20 min in a JS.4.2 rotor (Beckman). For lysis, cell pellets were resuspended in 80 ml of lysis buffer (20 mM Tris-HCl pH 8.5, 10% glycerol 0.5 mM EDTA, 500 mM KCl, Roche protease inhibitor tablets and 2 mM tris(2-carboxyethyl)phosphine (TCEP)) + 0.7 mM PMSF. The lysate was sonicated for 120 s (5 s on, 5 s off) at 40% on a Sonics Vibra-Cell sonicator. Insoluble material was removed by centrifugation at 20,000 rpm for 30 min 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 12 ml of 1× BXT (IBA) supplemented with 10% glycerol and 1 mM DTT. The MH-containing fractions were pooled and loaded onto a HiLoad 16/600 Superdex 75 equilibrated in gel filtration buffer (20 mM Tris-HCl pH 8.5, 10% glycerol 0.5 mM EDTA, 100 mM KCl and 0.5 mM TCEP). MH-containing fractions were pooled, aliquoted and snap-frozen in liquid N2. About 36 mg 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.
Preparation and purification of chromatinized origin DNA
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 (25 mM HEPES-KOH pH 7.6, 80 mM KCl, 100 mM sodium acetate, 0.5 mM 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 4 °C. Reconstitution conditions were optimized by small-scale titration and nucleosomes checked by 6% native PAGE.
Preparation and purification of MH-capped origin DNA
Short 168-bp MH-flanked origins
The conjugation of MH with origin substrates was performed in 50 mM Tris-HCl pH 8.0, 1 mM EDTA and 0.5 mM 2-mercaptoethanol supplemented with 100 µM S-adenosylmethionine (NEB). The reaction was carried out overnight at 30 °C, with a 10:1 molar ratio of MH:DNA. After conjugation, reactions were centrifuged at 14,680 rpm for 5 min and loaded onto a 1 ml RESOURCE-Q column equilibrated into DNA buffer (50 mM Tris-HCl pH 8.0 and 5 mM 2-mercaptoethanol). MH-conjugated DNA was eluted in a linear gradient of DNA buffer B (50 mM Tris-HCl pH 8.0, 5 mM 2-mercaptoethanol and 2 M NaCl) over 24 column volumes. Fractions containing MH-conjugated DNA were pooled, concentrated and stored at −80 °C. Conjugations were checked by 6% native PAGE.
6× ARS1 MH-flanked array
The conjugation of MH with origin substrates was performed in 25 mM Tris-HCl pH 7.5, 10 mM magnesium acetate, 50 mM potassium acetate and 1 mg ml−1 BSA supplemented with 150 µM S-adenosylmethionine (NEB). The reaction was carried out at 32 °C for 1 h then overnight at 4 °C, with a 20:1 molar ratio of MH:DNA. After conjugation, reactions were centrifuged at 14,680 rpm for 5 min and loaded onto a Superose 6 Increase 10/300 column equilibrated into array buffer (25 mM HEPES-KOH pH 7.5, 200 mM NaCl and 1 mM DTT). Fractions containing MH-conjugated array DNA were pooled, concentrated and stored at 4 °C. Conjugations were checked by 6% native PAGE.
616-bp ARS1 circles
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 30 min at 37 °C followed by enzyme inactivation at 80 °C for 2 min. After the ligation step, the DNA was concentrated as described and incubated with T5 exonuclease (NEB; 37 °C for 1 h) 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).
In vitro CMG assembly on short chromatinized origins
ARS1 nucleosome-flanked origin DNA (20 nM) was incubated with 52 nM ORC, 52 nM Cdc6 and 110 nM Mcm2–7–Cdt1 for 30 min at 24 °C in loading buffer (25 mM HEPES-KOH pH 7.6, 100 mM potassium glutamate, 10 mM magnesium acetate, 0.02% NP-40 and 0.5 mM TCEP) + 5 mM ATP. The reaction was supplemented with 80 nM DDK, and incubation continued for a further 10 min at 24 °C. Nucleoprotein complexes were isolated by incubation with 5 µl MagStrep ʻtype3̓ XT beads (IBA) pre-washed in 1× loading buffer for 30 min at 24 °C. The beads were washed three times with 100 µl wash buffer (25 mM HEPES-KOH pH 7.6, 105 mM potassium glutamate, 5 mM magnesium acetate, 0.02% NP-40 and 500 mM NaCl) and once with 100 µl loading buffer. Loaded, phosphorylated double hexamers were eluted in 20 μl elution buffer (25 mM HEPES-KOH pH 7.6, 105 mM potassium glutamate, 10 mM magnesium acetate, 0.02% NP-40, 0.5 mM TCEP, 27 mM biotin and 5 mM ATP) for 10 min at 24 °C. The remaining supernatant was removed and incubated with 200 nM CDK for 5 min at 30 °C. A mix of firing factors was then added to a final concentration of 30 nM Dpb11, 100 nM GINS, 80 nM Cdc45, 20 nM Pol ε, 30 nM Sld3–Sld7 and 50 nM Sld2. After 30 min of incubation, the reaction was applied directly to grids or diluted fivefold in 1× loading buffer for ReconSil experiments.
In vitro CMG assembly on 6× ARS1 MH-capped array
MH-capped ARS1 array DNA (5 nM) was incubated with 52 nM ORC, 52 nM Cdc6 and 110 nM Mcm2–7–Cdt1 for 30 min at 24 °C in loading buffer (25 mM HEPES-KOH pH 7.6, 100 mM potassium glutamate, 10 mM magnesium acetate, 0.02% NP-40 and 0.5 mM TCEP) + 5 mM ATP. The reaction was supplemented with 80 nM DDK, and incubation continued for a further 10 min at 24 °C. Nucleoprotein complexes were isolated by incubation with 5 µl MagStrep ʻtype3̓ XT beads (IBA) pre-washed in 1× loading buffer for 30 min at 24 °C. The beads were washed three times with 100 µl wash buffer (25 mM HEPES-KOH pH 7.6, 105 mM potassium glutamate, 5 mM magnesium acetate, 0.02% NP-40 and 500 mM NaCl) and once with 100 µl loading buffer. Loaded, phosphorylated double hexamers were eluted in 20 μl elution buffer (25 mM HEPES-KOH pH 7.6, 105 mM potassium glutamate, 10 mM magnesium acetate, 0.02% NP-40, 0.5 mM TCEP, 27 mM biotin and 5 mM ATP) for 10 min at 24 °C. The remaining supernatant was removed and incubated with 200 nM CDK for 5 min at 30 °C. A mix of firing factors was then added to a final concentration of 90 nM Dpb11, 300 nM GINS, 240 nM Cdc45, 60 nM Pol ε, 90 nM Sld3–Sld7 and 150 nM Sld2. After 30 min 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 0.1 U diluted in 1× loading buffer. Incubation was performed for 10 min at 30 °C before applying to EM grids.
In vitro DNA replication assays
Replication assays were performed as described previously52. The reactions were incubated in a ThermoMixer at 30 °C with 1,250 rpm shaking. The reaction buffer was as follows: 25 mM HEPES-KOH pH 7.6, 10 mM magnesium acetate, 2 mM DTT, 0.02% NP-40, 100 mM potassium glutamate and 5 mM ATP. MCM helicase loading reaction (5 µl) contained 30 nM ORC, 30 nM Cdc6, 60 nM Mcm2–7–Cdt1 (or MCM mutants) and either 4 nM ARS-containing 10.6 kb supercoiled plasmid (pJY22; Supplementary Table 2) or 40 nM ARS-containing short linear DNA (flanked by nucleosomes or MH; Supplementary Table 2) as for Fig. 1. After 20 min, DDK was added to a final concentration of 50 nM and further incubated for 20 min. Next, the reaction volume was doubled (final volume was 10 µl) by adding proteins (20 nM Pol ε, 30 nM Dpb11, 20 nM GINS, 50 nM Cdc45, 20 nM CDK, 50 nM RPA, 10 nM TopoI, 100 nM Pol α, 25 nM Sld3–Sld7, 10 nM Mcm10 and 50 nM Sld2) and nucleotides (200 µM CTP, 200 µM GTP, 200 µM UTP, 80 µM dCTP, 80 µM dGTP, 80 µM dTTP, 80 µM dATP and 50 nM α32P-dCTP). For replication reactions with linear DNA (Fig. 1) Pol ε exo- was used instead of Pol ε wild type to reduce end labelling and the concentration of deoxynucleotides was modified (that is, 30 µM dCTP, 30 µM dGTP, 30 µM dTTP, 30 µM dATP and 100 nM α32P-dCTP). The reactions were stopped by EDTA after 15 and 30 min for reactions with 10.6-kb supercoiled DNA or after 20 min for reactions with short linear DNA substrates and processed as described51,52. The replication products were separated using 0.8% agarose alkaline gel for 17 h at 25 V for reactions with 10.6-kb supercoiled DNA. For reactions with short DNA substrates, samples were separated using 2% agarose alkaline gel for 4 h at 38 V. The image signal from Fig. 1e was background-subtracted in Fiji using the subtract background algorithm in Fiji v.2.0.0 (ref. 53).
DNA topology assay
The experiment was performed as described previously1. The concentrations of proteins were as follows: 10 nM ORC, 50 nM Cdc6, 100 nM Mcm2–7–Cdt1 (or Mcm mutants), 80 nM DDK for the helicase loading step (5 µl) and 20 nM Pol ε, 30 nM Dpb11, 40 nM GINS, 50 nM Cdc45, 30 nM CDK, 10 nM TopoI, 25 nM Sld3–7, 5 nM Mcm10, 50 nM Sld2 for the helicase activation step (10 µl). Radiolabelled 616-bp circular DNA (25 fmol) 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 21 h at 90 V using Protean II XL Cell apparatus (Bio-Rad) at room temperature. The 0.7-mm gel was dried (without fixation) at 80 °C for 105 min, exposed to a phosphor screen and scanned with the use of Typhoon phosphor imager.
Sample preparation and data collection for NS-EM
NS-EM sample preparation was performed on 400-mesh copper grids with carbon film (Agar Scientific). Grids were glow-discharged for 30 s at 45 mA using a K100X glow discharge unit (Electron Microscopy Sciences) before a 4-µl sample was applied to the grids and incubated for 2 min. Grids were stained by two successive applications of 4 µl 2% (w/v) uranyl acetate with blotting between the first and second application. Stained grids were blotted after 20 s to remove excess stain. Unless described otherwise, data collection was carried out on a Tecnai LaB6 G2 Spirit transmission electron microscope (FEI) operating at 120 keV. A 2K × 2K GATAN Ultrascan 100 camera was used to collect micrographs at a nominal magnification of 30,000 (with a physical pixel size of 3.45 Å per pixel) within a −0.5 to −2.0 µm defocus range.
NS-EM image processing
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.
ReconSil image processing
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 yielded signal-enhanced ‘ReconSiled’ micrographs reconstituting the context of complete origins of replication. ReconSiled micrographs were used for the selection and rejection of origin nucleoproteins for further analysis.
ReconSil data analysis and statistics
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.
Sample preparation and data collection for cryo-EM
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 1 min at 45 mA using a K100X glow discharge unit (Electron Microscopy Sciences) before plunge freezing. Samples were prepared by applying 4 µl of undiluted CMG assembly reactions for 2 min on a grid equilibrated to 25 °C in 90% humidity. The grid was blotted for 4.5 s and plunged into liquid ethane. Data collection was performed on an in-house Thermo Fisher Scientific Titan Krios transmission electron microscope operated at 300 kV, 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 Å per pixel, with a total electron dose of 51.4 electrons per Å2 during a total exposure time of 10 s dose-fractionated into 32 movie frames (Extended Data Table 1). We used a slit width of 20 eV on the energy filter and a defocus range of −2.0 to −4.4 μm. A total of 65,286 micrographs were collected from two separate sessions.
Cryo-EM image processing
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 ε on both CMG molecules were un-binned and refined to yield maps with resolutions of 7.7 and 14.4 Å. 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 Å (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-Å 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-Å resolution. To better resolve the DNA inside the MCM central channel, densities corresponding to Cdc45, GINS and Pol ε 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-Å 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-Å 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.
Model building and refinement
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.
Map and model visualization
Maps were visualized in UCSF Chimera66 and ChimeraX67 and all model illustrations and morphs were prepared using ChimeraX or PyMOL.
Statistics and reproducibility
Statistical analysis was performed using a two-tailed Welch̓s 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 the Nature Research Reporting Summary linked to this paper.
We wish to thank the author of this article for this remarkable content
Mechanism of replication origin melting nucleated by CMG helicase assembly – Nature
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