Protein expression and purification<\/p>\n
HpaII methyltransferase\u00a0(MH), ORC, Cdc6, Mcm2\u20137\u2013Cdt1, DDK, CDK, (yeast-expressed) Sld3\/7, Cdc45, GINS, (yeast-expressed) Pol \u03b5, Mcm10, RPA, topoisomerase I (TopoI), Pol \u03b1 and Rad53 were expressed and purified as previously described24<\/a>,30<\/a>,40<\/a>,46<\/a>,66<\/a>,67<\/a>,68<\/a>,69<\/a>. All mutant constructs were expressed and purified following the same protocol as was used for the wild-type protein unless stated otherwise. All buffers described below are also reported in Supplementary Table 1<\/a>.<\/p>\n Cell lines<\/p>\n Sf21 insect cells were obtained in-house from the Cell Services Science and Technology Platform. These cells were not authenticated and tested negative for mycoplasma contamination.<\/p>\n Cloning, expression and purification of Twin-Strep-tagged Sld3\/7<\/p>\n Codon-optimized gene blocks (IDT) encoding Saccharomyces cerevisiae Sld3 in-frame with a tobacco etch virus (TEV) protease cleavage site and a C-terminal Twin-Strep-tag (TST) as well as S. cerevisiae Sld7 were inserted into GoldenBac shuttle vectors pGB-01;02 and pGB-02;03. Subsequently, Sld3-TEV-TST and Sld7 expression cassettes were subcloned into pGB-dest using GoldenBac assembly70<\/a> and transformed into electrocompetent EMBacY cells (Geneva Biotech). Cells were screened by blue\u2013white selection for successful bacmid integration and selected colonies were grown overnight at 37\u2009\u00b0C. Cells were collected by centrifugation and bacmids were purified by isopropanol precipitation. A total of 45\u2009\u00b5g bacmid DNA was mixed thoroughly with 13.5\u2009\u00b5l FuGENE HD Transfection Reagent (Promega) and 450\u2009\u00b5l Sf-900 III SFM medium and incubated for 30\u2009min at room temperature. Two hundred microlitres of transfection mix was added dropwise to 2\u2009ml Sf21 insect cells seeded at 0.5\u2009million cells per ml in a six-well plate. Plates were incubated for 3\u20135 days at 27\u2009\u00b0C in a wet-towel box. Efficient transfection was monitored through YFP fluorescence. Adherent cells were resuspended and supernatant containing P0 baculovirus was collected. To increase the multiplicity of infection, 46\u2009ml Sf21 insect cells at 0.5\u2009million cells per ml were inoculated with 4\u2009ml P0 baculovirus in suspension and cultured at 27\u2009\u00b0C shaking at 120\u2009rpm. After viability had dropped below 90%, cells were pelleted by centrifugation at 380g for 15\u2009min at 4\u2009\u00b0C, and supernatant containing the amplified P1 virus was sterile-filtered (0.22\u2009\u00b5m pore size) and stored at 4\u2009\u00b0C. One litre of Sf21 insect cells were seeded at one\u2009million cells per ml in Sf-900 III SFM medium and infected with 0.5 % v\/v P1 virus. Cells were collected 48\u2009h after baculovirus-induced cell-cycle arrest by centrifugation for 15\u2009min at 180g at 4\u00b0\u2009C, and pellets were flash-frozen in liquid nitrogen and stored at \u221280\u2009\u00b0C.<\/p>\n Cells were thawed and resuspended in 50\u2009ml buffer A (25\u2009mM HEPES-KOH pH 7.5, 500\u2009mM KCl, 10% v\/v glycerol, 0.02% w\/v NP-40, 1\u2009mM EDTA and 1\u2009mM DTT) supplemented with one cOmplete EDTA-free protease inhibitor tablet (Merck) and 0.7\u2009mM phenylmethylsulfonyl fluoride (PMSF), then lysed by sonication on ice for 2\u2009min (1-s pulse-on, 4-s pulse-off). The lysate was clarified by ultracentrifugation for 1\u2009h at 45,000\u2009rpm in a Ti45 rotor (Beckman) at 4\u2009\u00b0C, and the supernatant was mixed with 2.4\u2009ml Bio-Lock (IBA) reagent and applied onto 1\u2009ml pre-equilibrated Strep-Tactin XT Superflow HighCapacity resin in a gravity column. The resin was washed with 100\u2009ml buffer A and 10\u2009mL buffer A supplemented with 2\u2009mM ATP and 10\u2009mM MgCl2. Protein was eluted with 10\u2009ml buffer A supplemented with buffer BXT (IBA). The eluate was pooled, concentrated and loaded onto a Superdex 200 Increase 10\/300 GL column (Cytiva) equilibrated in buffer A. Gel-filtered Sld3\/7 was concentrated to approximately 1.5\u2009mg\u2009ml\u22121, aliquoted and flash-frozen in liquid nitrogen.<\/p>\n Cloning, expression and purification of Dpb11<\/p>\n Codon-optimized S. cerevisiae Dpb11 followed by a 3C protease cleavage site and a C-terminal 3\u00d7Flag tag was subcloned into a pGB-04;05 shuttle vector and subsequently transformed into electrocompetent EMBacY cells. Bacmid and baculoviruses were prepared as described above; 1\u2009l Sf21 insect cells at one\u2009million cells per ml were infected with 0.5% v\/v P1 virus and collected 48\u2009h after cell-cycle arrest.<\/p>\n The cell pellet was resuspended in 50\u2009mL buffer A supplemented with one cOmplete EDTA-free protease inhibitor tablet (Merck) and 0.7\u2009mM PMSF, lysed by sonication on ice for 2\u2009min (1-s pulse-on, 4-s pulse-off) and ultracentrifuged at 45,000\u2009rpm at 4\u2009\u00b0C for 1\u2009h. The soluble phase was mixed with 2.4\u2009ml Bio-Lock Reagent and passed through 1\u2009ml pre-equilibrated anti-Flag M2 Affinity Gel (Sigma) in a gravity column. The column was washed with 150\u2009ml buffer A and 10\u2009ml buffer A supplemented with 2\u2009mM ATP and 10\u2009mM MgCl2. To elute bead-bound protein, the beads were resuspended in 5\u2009ml buffer A supplemented with 0.5\u2009mg\u2009ml\u22121 3\u00d7Flag peptide and incubated for 5\u2009min, after which the flow-through was collected. The eluate was diluted to 150\u2009mM KCl and loaded onto a 1\u2009ml Mono S 5\/50 column (Cytiva) equilibrated in buffer B (25\u2009mM HEPES-KOH pH 7.5, 150\u2009mM KCl, 10% v\/v glycerol, 0.02% w\/v NP-40, 1\u2009mM EDTA and 1\u2009mM DTT). After washing the column with 10\u2009ml buffer B, Dpb11 was eluted with a linear gradient of 150\u20131,000\u2009mM KCl in buffer B over 20 column volumes. Fractions containing pure Dpb11 were pooled and dialysed against buffer C (25\u2009mM HEPES-KOH pH 7.5, 300\u2009mM KOAc, 10% v\/v glycerol, 0.02% NP-40, 1\u2009mM EDTA and 1\u2009mM DTT) at 4\u2009\u00b0C overnight while stirring. Subsequently, Dpb11 was concentrated to approximately 0.5\u2009mg\u2009ml\u22122, aliquoted and flash-frozen in liquid nitrogen.<\/p>\n Dpb11 mutants containing charge-reversal substitutions (Dpb11(3E), Dpb11(3X) and Dpb11(3E\/3X)) were purified using gel filtration instead of cation-exchange chromatography. The reason for this alteration was the predicted isoelectric point of the mutant proteins, which matches the pH of buffers B and C. After Flag affinity purification, the eluate was concentrated and loaded onto a Superdex 200 Increase 10\/300 GL column equilibrated in buffer C. Dpb11-containing fractions were pooled, concentrated to approximately 0.5\u2009mg\u2009ml\u22121, aliquoted and flash-frozen in liquid nitrogen.<\/p>\n Cloning, expression and purification of Sld2<\/p>\n An expression cassette encoding S. cerevisiae Sld2 in-frame with an N-terminal VNp6 peptide tag71<\/a> and a retro-protein XXA solubility tag72<\/a>, as well as a C-terminal Twin-Strep-tag, was subcloned into a pET303 backbone and transformed into bacterial T7 Express cells. Multiple colonies were picked to inoculate 4\u00d7 1\u2009l LB\u2009+\u2009100\u2009\u00b5g\u2009ml\u22121 carbenicillin and incubated static overnight at 37\u2009\u00b0C. The next morning, cultures were transferred to 30\u2009\u00b0C and grown to an optical density at 600\u2009nm (OD600\u2009nm) of 0.8, shaking at 200\u2009rpm. Isopropyl \u03b2-d-1-thiogalactopyranoside (IPTG; 80\u2009\u00b5M) was added to each flask to induce the expression of Sld2 for 21\u2009h at 30\u2009\u00b0C at 200\u2009rpm. Subsequently, cells were pelleted by centrifugation at 4,000g for 10\u2009min at 4\u2009\u00b0C, flash-frozen and stored at \u221280\u2009\u00b0C.<\/p>\n Cells were thawed and resuspended in 100\u2009ml buffer D (25\u2009mM HEPES-KOH pH 7.5, 800\u2009mM KCl, 10% v\/v glycerol, 1\u2009M sorbitol, 2\u2009mM ATP, 10\u2009mM MgCl2, 0.02% v\/v NP-40, 0.1% w\/v Tween-20, 1\u2009mM DTT) with two cOmplete EDTA-free protease inhibitor tablets (Merck) and 0.7\u2009mM PMSF, then sonicated on ice for 2\u2009min (5-s pulse-on, 5-s pulse-off). The lysate was clarified by centrifugation in a JA-25.50 rotor at 18,000\u2009rpm for 20\u2009min at 4\u2009\u00b0C, and the supernatant was applied onto a gravity column packed with 1\u2009ml Strep-Tactin XT Superflow High Capacity Resin equilibrated in buffer D. The resin was washed with 75\u2009ml buffer D followed by 25\u2009ml buffer E (25\u2009mM HEPES-KOH pH 7.5, 500\u2009mM NaCl, 10% v\/v glycerol, 0.02% w\/v NP-40, 1\u2009mM EDTA and 1\u2009mM DTT), after which Sld2 was eluted by passing 10\u00d7 1\u2009ml buffer E supplemented with buffer BXT through the resin. The highest-concentration fraction was identified by SDS\u2013PAGE (Coomassie staining) and dialysed in buffer F (25\u2009mM HEPES-KOH pH 7.5, 700\u2009mM KOAc, 40% v\/v glycerol, 0.02% w\/v NP-40, 1\u2009mM EDTA and 1\u2009mM DTT) for 4\u2009h at 4\u2009\u00b0C. Sld2 was aliquoted at approximately 0.8\u2009mg\u2009ml\u22121 and flash-frozen in liquid nitrogen. For the phosphomimetic Sld2(8D) variant, aspartate substitutions were introduced at the following residues: threonine 84, serine 100, serine 128, serine 138, threonine 168, serine 172, serine 188 and serine 208.<\/p>\n Cloning, expression and purification of ALFA-tagged Pol \u03b5<\/p>\n Codon-optimized S. cerevisiae Pol2-3\u00d7Flag, Dpb2, Dpb3 and Dpb4-ALFA were subcloned into GoldenBac shuttle vectors and assembled into a co-expression plasmid, pGB-dest-PolE, as described above for Sld3\/7. Similarly, electrocompetent EMBacY cells were transformed with pGB-dest-PolE to prepare bacmids and generate a P1 baculovirus as described above. One\u2009billion Sf21 insect cells were seeded in 1\u2009l Sf-900 III SFM medium and infected with 0.5% v\/v P1 baculovirus, incubated at 27\u2009\u00b0C at 120\u2009rpm and collected 48\u2009h after cell-cycle arrest by centrifugation at 180g at 4\u2009\u00b0C for 15\u2009min. The cell pellets were flash-frozen in liquid nitrogen and stored at \u221280\u2009\u00b0C. To purify ALFA-tagged Pol \u03b5, the cell pellets were resuspended in 50\u2009ml buffer G (25\u2009mM HEPES-KOH pH 7.6, 400\u2009mM KOAc, 10% v\/v glycerol and 2\u2009mM DTT) supplemented with one cOmplete EDTA-free protease inhibitor tablet (Merck), and lysed by sonication on ice for 2\u2009min (1-s pulse-on, 4-s pulse-off). The lysate was clarified by ultracentrifugation at 45,000\u2009rpm for 45\u2009min at 4\u2009\u00b0C in a Ti45 rotor, and the supernatant was passed twice through a column packed with 1\u2009ml anti-Flag M2 affinity gel equilibrated in buffer G. The column was washed with 150\u2009ml buffer G and 20\u2009ml buffer G\u2009+\u20092\u2009mM ATP and 10\u2009mM Mg(OAc)2. Protein was eluted by incubating the resin three times in 5\u2009ml buffer G\u2009+\u20090.5\u2009mg\u2009ml\u22121 3\u00d7 Flag peptide for 5\u2009min and collecting the flow-through. The eluate was loaded onto a 5-ml Heparin HP column (Cytiva) and eluted over 25 column volumes with a linear gradient of 400\u20131,000\u2009mM KOAc in buffer G. Fractions containing Pol \u03b5 were concentrated and gel-filtered onto a HiLoad 16\/60 Superdex 200-pg column (Cytiva). Pol \u03b5 was concentrated to approximately 1\u2009mg\u2009ml\u22121, aliquoted and flash-frozen in liquid nitrogen.<\/p>\n Cloning, expression and purification of Sic1<\/p>\n T7 Express cells (NEB) were transformed with hexahistidine-tagged S. cerevisiae Sic173<\/a>. Transformant colonies were incubated overnight in 100\u2009ml LB supplemented with 100\u2009\u00b5g\u2009ml\u22121 carbenicillin at 37\u2009\u00b0C shaking at 200\u2009rpm. Two litres of LB medium and 100\u2009\u00b5g\u2009ml\u22121 carbenicillin were inoculated with 1% v\/v dense overnight culture and grown to an OD600\u2009nm of 0.6 at 37\u2009\u00b0C and 200\u2009rpm. Expression of Sic1 was induced by adding 0.5\u2009mM IPTG, after which the cultures were incubated for 3\u2009h at 37\u2009\u00b0C at 200\u2009rpm. Cells were collected by centrifugation at 4,000g for 10\u2009min at 4\u2009\u00b0C, and the cell pellet was flash-frozen in liquid nitrogen and stored at \u221280\u2009\u00b0C.<\/p>\n The pellet was dissolved in 100\u2009ml buffer H (25\u2009mM HEPES-KOH pH 7.5, 500\u2009mM NaCl, 10% v\/v glycerol, 1\u2009mM EGTA, 0.2% w\/v Triton X-100, 0.5\u2009mM TCEP, 10\u2009mM Imidazole) with two cOmplete EDTA-free protease inhibitor tablets (Merck) and 0.7\u2009mM PMSF in a beaker. Lysozyme (0.2\u2009mg\u2009ml\u22121) was added and incubated with the cell suspension for 10\u2009min while stirring. Cells were then sonicated on ice for 5\u2009min (2-s pulse-on, 5-s pulse-off), and the debris was removed by centrifugation at 20,000\u2009rpm in a JA-25.50 rotor for 30\u2009min at 4\u2009\u00b0C. Two millilitres of Ni-NTA beads (QUIAGEN) were equilibrated in buffer H and rotated with the clarified lysate for 1\u2009h at 4\u2009\u00b0C. Subsequently, beads were collected in a gravity column and washed with 150\u2009ml buffer H and 20\u2009ml buffer H supplemented with 2\u2009mM ATP and 10\u2009mM MgCl2. Protein was eluted with 15\u2009ml buffer H supplemented with 250\u2009mM imidazole, concentrated and gel-filtered on a HiLoad 16\/60 Superdex 75-pg column (Cytiva) equilibrated in buffer I (25\u2009mM HEPES-KOH pH 7.5, 5% v\/v glycerol, 5\u2009mM MgCl2, 0.5\u2009mM EDTA and 0.5\u2009mM TCEP) supplemented with 200\u2009mM NaCl. Gel filtration did not yield pure Sic1. Consequently, peak fractions were loaded onto a 1-ml Mono S column (Cytiva) and washed with 10 column volumes of buffer I supplemented with 50\u2009mM NaCl. Sic1 was eluted with a linear gradient of 50\u20131,000\u2009mM NaCl in buffer I over 30 column volumes, and fractions containing pure Sic1 were dialysed at 4\u00b0\u2009C in buffer I\u2009+\u2009200\u2009mM NaCl for 3\u2009h under agitation. Sic1 was concentrated to 10.8\u2009mg\u2009ml\u22121 and flash-frozen in liquid nitrogen.<\/p>\n Cloning, expression and purification of Twin-Strep-tagged SUMO-Mcm10<\/p>\n S. cerevisiae Mcm10 was cloned in-frame with an N-terminal 10\u00d7His-SUMO cassette and a C-terminal Twin-Strep-tag, and transformed into Rosetta 2 pLysS cells. Multiple colonies were picked and grown overnight at 37\u2009\u00b0C in 100\u2009ml LB supplemented with 100\u2009\u00b5g\u2009ml\u22121 carbenicillin and 33\u2009\u00b5g\u2009ml\u22121 chloramphenicol. Then, 6\u00d7 1\u2009l LB supplemented with 100\u2009\u00b5g\u2009ml\u22121 carbenicillin and 33\u2009\u00b5g\u2009ml\u22121 chloramphenicol were each inoculated with 10\u2009ml dense overnight culture and grown to an OD600\u2009nm of 0.7 at 37\u2009\u00b0C and 200\u2009rpm. Overexpression was induced by the addition of 0.5\u2009mM IPTG and continued for 16\u2009h at 16\u2009\u00b0C. Cells were collected by centrifugation at 4,000g for 10\u2009min at 4\u2009\u00b0C, flash-frozen in liquid nitrogen and stored at \u221280\u2009\u00b0C. The cell pellet was resuspended in 230\u2009ml buffer J (25\u2009mM HEPES-KOH pH 7.6, 500\u2009mM NaCl, 10% v\/v glycerol, 1\u2009mM EDTA, 0.05% w\/v Tween-20 and 1\u2009mM DTT) supplemented with four cOmplete EDTA-free protease inhibitor tablets (Merck), then lysed by sonication on ice for 5\u2009min (2-s pulse-on, 5-s pulse-off). The lysate was clarified by centrifugation in a JA-25.50 rotor at 20,000\u2009rpm for 30\u2009min at 4\u2009\u00b0C, and the supernatant was loaded onto a 1-ml cOmplete His-Tag Purification column (Merck) installed in tandem with a 1-ml Strep-Tactin XT 4Flow High Capacity column (IBA), both pre-equilibrated in buffer J. The columns were washed with buffer J until the absorbance at 280\u2009nm returned to its baseline signal, after which protein was eluted from the His-Tag Purification column into the connected Strep-Tactin XT column with 9\u2009ml buffer J supplemented with 200\u2009mM imidazole. The His-Tag Purification column was disconnected, the Strep-Tactin XT column was washed with 9\u2009ml buffer B, and this was finally followed by elution with 9\u2009ml buffer K (25\u2009mM HEPES pH 7.6, 300\u2009mM NaCl, 10% v\/v glycerol, 0.05% w\/v Tween-20, 1\u2009mM DTT and 5\u2009mM desthiobiotin). Mcm10-containing fractions were pooled and dialysed overnight into buffer L (25\u2009mM HEPES pH 7.6, 200\u2009mM NaCl, 20% v\/v glycerol, 0.05% w\/v Tween-20, 1\u2009mM EDTA and 2\u2009mM DTT) at 4\u2009\u00b0C while stirring. Mcm10 was aliquoted at a concentration of approximately 0.5\u2009mg\u2009ml\u22121 and flash-frozen in liquid nitrogen.<\/p>\n Preparation of an MH-conjugated ARS1 DNA template<\/p>\n A 168-bp DNA template containing the S. cerevisiae origin of replication ARS1, flanked by two MH recognition sites, was generated by PCR and purified as previously described74<\/a>,75<\/a>. The DNA template was covalently tethered to either Twin-Strep-tagged MH or tandem ALFA\/Twin-Strep-tagged MH using previously established protocols30<\/a>.<\/p>\n S-CDK prephosphorylation of Sld3\/7<\/p>\n Sld3\/7 prephosphorylation by S-CDK was modified from previous protocols24<\/a>. In brief, 900\u2009nM Twin-Strep-tagged Sld3\/7 was phosphorylated by 100\u2009nM S-CDK in buffer M (40\u2009mM HEPES-KOH pH 7.5, 310 mM potassium glutamate, 10\u2009mM Mg(OAc)2, 10% v\/v glycerol, 0.02% w\/v NP-40, 1\u2009mM DTT, 2\u2009mM ATP and 0.4\u2009mg\u2009ml\u22121 BSA) in a total volume of 100\u2009\u00b5l for 8\u2009min at 24\u2009\u00b0C and 1,250\u2009rpm. Phosphorylation was stopped by adding 2.2\u2009\u00b5M Sic1 to the reaction and incubating it for 2\u2009min at 24\u2009\u00b0C and 1,250\u2009rpm. Then, 500\u2009mM KCl was added to the reactions, which were then bound to 10\u2009\u00b5l MagStrep \u2018type3\u2019 XT slurry (IBA) equilibrated in buffer N (25\u2009mM HEPES-KOH pH 7.5, 500\u2009mM KCl, 5\u2009mM Mg(OAc)2, 10% v\/v glycerol, 0.02% w\/v NP-40 and 1\u2009mM DTT). After 30\u2009min of incubation at 24\u2009\u00b0C and 1,250\u2009rpm, beads were washed five times with 200\u2009\u00b5l buffer K, and ppSld3\/7 was eluted in 10\u2009\u00b5l buffer N\u2009+\u200925\u2009mM d-biotin for 10\u2009min at 24\u2009\u00b0C and 1,250\u2009rpm, aliquoted, flash-frozen in liquid nitrogen and stored at \u221280\u2009\u00b0C. Successful phosphorylation and approximate yield were estimated by SDS\u2013PAGE. For cryo-EM experiments, ppSld3\/7 was prepared in parallel and not flash-frozen before use.<\/p>\n Rad53 prephosphorylation of Sld3\/7<\/p>\n Rad53-prephosphorylated Sld3\/7 was prepared by incubating 700\u2009nM Twin-Strep-tagged Sld3\/7 with 356\u2009nM Rad53 in 100\u2009\u00b5L buffer M for 30\u2009minutes at 30\u2009\u00b0C and 1,250\u2009rpm. Subsequently, Rad53-prephosphorylated Sld3\/7 was purified by Strep-Tactin XT affinity purification exactly as described above for CDK-prephosphorylated Sld3\/7.<\/p>\n In vitro ATP\u2013dCMGE assembly<\/p>\n ATP-bound dCMGE complexes were assembled as previously described30<\/a>,47<\/a> with minor modifications. First, MCM-DHs were loaded for 30\u2009min at 30\u00b0\u2009C and 1,250\u2009rpm by co-incubating 20\u2009nM MH-conjugated ARS1 with 52\u2009nM ORC, 52\u2009nM Cdc6 and 110\u2009nM Mcm2\u20137\u2013Cdt1 in 100\u2009\u00b5l buffer O (25\u2009mM HEPES-KOH pH 7.5, 100 mM potassium glutamate, 10\u2009mM Mg(OAc)2, 1\u2009mM ATP and 0.02% w\/v NP-40). Afterwards, loaded MCM-DHs were phosphorylated with 80\u2009nM DDK at 24\u2009\u00b0C and 1,250\u2009rpm for 10\u2009min, then bound for 30\u2009min at 24\u2009\u00b0C and 1,250\u2009rpm to 5\u2009\u00b5l MagStrep \u2018type3\u2019 XT slurry equilibrated in buffer P (25\u2009mM HEPES-KOH pH 7.5, 100 mM potassium glutamate, 10\u2009mM Mg(OAc)2 and 0.02% w\/v NP-40). The beads were washed three times with 200\u2009\u00b5l buffer Q (25\u2009mM HEPES-KOH pH 7.5, 500\u2009mM NaCl, 5\u2009mM Mg(OAc)2 and 0.02% w\/v NP-40) and once with 200\u2009\u00b5l buffer P, then eluted in 20\u2009\u00b5l buffer O\u2009+\u200925\u2009mM d-biotin for 15\u2009min at 24\u2009\u00b0C and 1,250\u2009rpm. S-CDK (200\u2009nM) was added to DDK-phosphorylated MCM-DHs, and dCMGE assembly was then started by further addition of 40\u2009nM Dpb11, 32.5\u2009nM Pol \u03b5, 133\u2009nM GINS, 106\u2009nM Cdc45, 40\u2009nM Sld3\/7 and 67\u2009nM Sld2. After 12\u2009min of incubation at 30\u2009\u00b0C and 1,250\u2009rpm, the reactions were immediately used for negative-stain grid preparation. Wild-type proteins were substituted with mutant constructs or omitted as specified. To split dCMGEs into sCMGEs, 100\u2009nM Mcm10 and 300\u2009nM RPA were added at the same time as the other firing factors.<\/p>\n In vitro pre-IC assembly<\/p>\n To assemble the pre-IC, DDK-phosphorylated MCM-DHs were prepared as described above, but eluted in the absence of ATP. Sld3\/7 and Sld2 were replaced by equimolar amounts of ppSld3\/7 and phosphomimetic Sld2(8D), and no CDK was added. DDK-phosphorylated DHs were incubated with ppSld3\/7, Sld2(8D), Dpb11, Pol \u03b5, GINS and Cdc45 in the absence of ATP and CDK for 10\u2009min at 24\u2009\u00b0C and 1,250\u2009rpm, after which reactions were either negatively stained, applied to cryo-EM grids and plunge-frozen using a Vitrobot, or further purified.<\/p>\n ALFA pull-down of pre-IC and dCMGE complexes<\/p>\n To purify either pre-IC or dCMGE, phospho-DH formation and respective complex assembly was done on an ALFA-tagged 2\u00d7MH-ARS1 DNA template. Twenty microlitres of assembly reaction prepared as described above was added to 15\u2009\u00b5l ALFA PE Selector slurry (NanoTag Biotechnologies) equilibrated in buffer P and bound for 5\u2009min at 24\u2009\u00b0C and 1,250\u2009rpm.<\/p>\n For pre-IC maturation reactions, beads were washed with 3\u00d7 150\u2009\u00b5l buffer P and eluted for 5\u2009min at 24\u00b0\u2009C and 1,250\u2009rpm in 20\u2009\u00b5l buffer P\u2009+\u2009200\u2009\u00b5M ALFA peptide (NanoTag Biotechnologies). Pre-IC to dCMGE conversion was triggered by adding 1\u2009mM ATP per reaction and analysed by nsEM.<\/p>\n To assay for the high-salt stability of the pre-IC and dCMGE, beads were washed with 3\u00d7 150\u2009\u00b5l of buffer R (25\u2009mM HEPES-KOH pH 7.5, 5\u2009mM Mg(OAc)2, 10% v\/v glycerol and 0.02% w\/v NP-40) and either 250\u2009mM potassium glutamate (low-salt wash) or 300\u2009mM KCl (high-salt wash). Elution was performed in 20\u2009\u00b5l buffer P\u2009+\u2009200\u2009\u00b5M ALFA peptide (no nucleotide for pre-IC reactions; 1\u2009mM ATP for dCMGE reactions) for 5\u2009min at 24\u2009\u00b0C and 1,250\u2009rpm.<\/p>\n In vitro DNA replication assay<\/p>\n DNA replication was performed at 30\u2009\u00b0C and 1,250\u2009rpm following previous protocols76<\/a>. In brief, MCM-DHs were loaded for 20\u2009min using 40\u2009nM ORC, 40\u2009nM Cdc6, 60\u2009nM Mcm2\u20137\u2013Cdt1 and 4\u2009nM 10.6\u2009kb pJY22 plasmid template DNA in buffer S (25\u2009mM HEPES-KOH pH 7.6, 100\u2009mM potassium glutamate, 10\u2009mM magnesium acetate, 5\u2009mM ATP, 0.02% NP-40-S and 2\u2009mM DTT). Loaded MCMs were phosphorylated with 50\u2009nM DDK for 15\u2009min. DNA replication was performed for 30\u2009min by adding final concentrations of 40\u2009nM Dpb11, 20\u2009nM Pol \u03b5, 20\u2009nM GINS, 80\u2009nM Cdc45, 20\u2009nM CDK, 25\u2009nM Sld3\/7, 50\u2009nM Sld2, 200\u2009nM RPA, 20\u2009nM TopoI, 50\u2009nM Pol \u03b1, 20\u2009nM Mcm10, 200\u2009\u03bcM each of CTP, GTP and UTP, 80\u2009\u03bcM dNTP and 33\u2009nM \u025132P-dCTP. When specified, Dpb11, Sld3\/7 and Sld2 proteins were excluded from reactions or substituted by mutant versions. In the CDK bypass experiment, reactions contained either 10\u2009nM wild-type Sld2 and 5\u2009nM Sld3\/7 or 50\u2009nM Sld2(8D) and 25\u2009nM phosphorylated Sld3\/7.<\/p>\n Reactions were stopped with 85\u2009mM EDTA, cleared over an Illustra MicroSpin G-50 column, denatured in 2% sucrose, 0.02% bromophenol blue, 60\u2009mM NaOH and 10\u2009mM EDTA, separated on 0.8% agarose gels in alkaline conditions containing 30\u2009mM NaOH and 2\u2009mM EDTA at approximately 1\u2009V per cm for 17\u2009h, fixed in cold 5% trichloroacetic acid, dried, exposed to phosphor screens and scanned using a Typhoon phosphor imager.<\/p>\n Sample preparation and data collection for nsEM<\/p>\n Carbon-coated 300-mesh copper grids (EM Resolutions) were glow-discharged at 25\u2009mA for 1\u2009min in a GloQube Plus (Quorum) in ambient air. Four-microlitre samples were incubated for 2\u2009min on a glow-discharged grid, blotted and negatively stained by two applications of 4\u2009\u00b5l 2% uranyl-acetate for 20\u2009s, after which the grid was blotted dry. nsEM micrographs were acquired using a Rio16 camera (Gatan Digital Micrograph) on a FEI Tecnai G2 Spirit Twin microscope operated at 120\u2009kV. Approximately 50\u2013150 micrographs were collected per dataset at 3.1\u2009\u00c5 per pixel (px) (29,000\u00d7 magnification) at \u22121 to \u22122 \u00b5m defocus.<\/p>\n nsEM image processing<\/p>\n Micrographs were imported in RELION-477<\/a> and contrast transfer function (CTF) was estimated using Gctf78<\/a>. MCM-containing particles were picked using crYOLO (v. 1.9.2)79<\/a>, imported into RELION and extracted with a 144-px box size. Extracted particles were 2D-classified in either RELION-4 or cryoSPARC (v.4.4.1)80<\/a>. Interpretable class averages were categorized (DH, pre-IC, cis- or trans-dCMGE, sCMGE) and particles were quantified. Complex assembly efficiency was calculated as the number of all detected target particles divided by the number of all licensed replication origins. For example, the number of pre-IC particles was divided by the sum of DHs and pre-IC particles. For Mcm10-dependent CMGE splitting experiments, all sCMGEs were multiplied by a factor of 0.5, to account for the fact that sCMGEs originate from a single, licensed replication origin.<\/p>\n Cryo-EM sample preparation for the pre-IC<\/p>\n MCM-DHs were loaded onto an ALFA-tagged 2\u00d7MH-ARS1 template and phosphorylated by DDK as described above. One hundred microlitres of DNA-loaded, phospho-DHs were purified on 15\u2009\u00b5l ALFA PE Selector beads for 10\u2009min at 24\u2009\u00b0C and 1,250\u2009rpm. Subsequently, beads were washed three times with 200\u2009\u00b5l buffer Q and once with 200\u2009\u00b5L buffer O and eluted for 10\u2009min in 20\u2009\u00b5l buffer O supplemented with 200\u2009\u00b5M ALFA peptide at 24\u2009\u00b0C and 1,250\u2009rpm. Given that ALFA elution yielded a higher number of phospho-DHs than Strep-Tactin XT purification did, the eluted phospho-DHs were incubated with a four times higher molar amount of firing factors (Dpb11, Pol \u03b5, GINS, Cdc45, ppSld3\/7 and Sld2(8D)) at 24\u2009\u00b0C and 1,250\u2009rpm for 10\u2009min.<\/p>\n Graphene-oxide-coated 300-mesh UltrAuFoil R1.2\/R1.3 grids were prepared on the day according to a previously published protocol47<\/a>. Four microlitres of sample was applied per grid in a Mark IV Vitrobot (FEI), followed by incubation for 60\u2009s at 24\u2009\u00b0C and 90% humidity. Grids were blotted for 3\u20134.5\u2009s at blot force 0 and plunge-frozen in liquid ethane.<\/p>\n Cryo-EM data collection for the pre-IC<\/p>\n A total of 59,347 movies were collected on a 300-kV FEI Titan Krios G3i at a nominal magnification of 130,000\u00d7 (0.95\u2009\u00c5 px\u22121 physical pixel size) using a Falcon IV direct electron detector in counting mode and a Selectris energy filter with a slit width of 10\u2009eV using EPU v.3.2. Three shots were acquired per hole at spot size 9 with a beam diameter of 660\u2009nm, a 100-\u00b5m objective aperture inserted and a defocus range from \u22122.0 to \u22123.0\u2009\u00b5m. Each movie was recorded with 1,674 electron event representation (EER) frames for 5.44\u2009s with a total fluence of 39 electrons per \u00c52 (Extended Data Table 1<\/a>).<\/p>\n Cryo-EM image processing for the pre-IC<\/p>\n A total of 59,347 EER movies were aligned and dose-weighted with 5\u2009\u00d7\u20095 patches using RELION\u2019s own implementation of MotionCor281<\/a>. Fifty-four internal frames were grouped into 31 fractions resulting in a dose per frame of 1.26 electrons per \u00c52. Motion-corrected micrographs were imported into cryoSPARC (v.4.4.1)80<\/a> and CTF estimation was done using Patch CTF. Initial particle picking was performed using Blob Picker with a diameter range of 200\u2013350\u2009\u00c5 and circular blobs. A total of 5,149,760 particles were extracted, fourfold binned to 3.8\u2009\u00c5 px\u22121 with a 150-px box size and cleaned up with multiple rounds of reference-free 2D classification with 400 classes and an uncertainty factor of 2. A total of 62,192 MCM-DH and pre-IC particles were selected from 2,867 micrographs containing more than 20 particles per micrograph in a defocus range from \u22121.5 to \u22122.5\u2009\u00b5m, and used to train a Topaz network with 75 expected particles per micrograph82<\/a>. After Topaz picking at an extraction threshold of \u22126 with an extraction radius of 28\u2009px, 1,572,464 particles were extracted and Fourier-cropped to 3.8\u2009\u00c5\u2009px\u22121 with a box size of 150\u2009px and subjected to 3 rounds of 2D classification yielding 302,557 DHs and 345,093 pre-ICs.<\/p>\n Ab-initio reconstructions of both DHs and pre-ICs were generated independently in C1. The initial volumes of each complex were used as 3D references for heterogenous refinement to further separate DHs and pre-ICs from each other and remove low-quality particles. A total of 279,730 DH particles were separated into 20 classes by alignment-free 3D classification in cryoSPARC. A total of 162,764 high-quality DH particles were selected, unbinned to 0.95\u2009\u00c5\u2009px\u22121 and refined to a final resolution of 2.8\u2009\u00c5 with C2 symmetry applied. After separating pre-IC particles from DH particles through heterogenous refinement, 335,896 binned pre-IC particles were non-uniform-refined with C2 symmetry applied, unbinned to 0.95\u2009\u00c5\u2009px\u22121 with a 600-px box size and 3D-classified without alignment, yielding 290,496 particles that were non-uniform-refined to 3.72\u2009\u00c5. This first unbinned reconstruction of the pre-IC was subjected to further classification in cryoSPARC to remove low-quality particles, resulting in a stack of 151,175 pre-IC particles exhibiting improved density quality. After homogenous refinement, symmetry expansion was performed. Local C1 refinement yielded a 3.4-\u00c5-resolution map of the pre-IC dimer, with some residual anisotropy visible. To improve the reconstruction further, refinement of a signal-subtracted monomer was performed. To generate the best possible mask for subtraction, a double-subtraction strategy was implemented. First, one asymmetric unit was locally refined using a soft mask around the top pre-IC monomer. The resulting, improved, reconstruction was used to generate a new soft mask for signal subtraction, which was used to accurately remove the signal from the top pre-IC monomer. The remaining pre-IC monomer was reconstructed and locally refined to 3.3-\u00c5 resolution, and showed improved isotropy. This map was used to generate a third soft mask for a second signal subtraction of the bottom monomer. This allowed us to determine the structure of the top monomer using local refinement to a resolution of 3.2\u2009\u00c5.<\/p>\n Cryo-EM sample preparation for phospho-DH-3745<\/p>\n DDK-phosphorylated, DNA-loaded MCM-DHs were eluted from MagStrep \u2018type3\u2019 XT beads in buffer T (25\u2009mM HEPES-KOH pH 7.5, 100\u2009mM KOAc, 0.02% w\/v NP-40 and 25\u2009mM d-biotin) and incubated with 32\u2009nM (yeast-expressed) Sld3\/7 and 127\u2009nM Cdc45 for 10\u2009min at 30\u2009\u00b0C and 1,250\u2009rpm. To stabilize the phospho-DH-3745 complex, the sample was cross-linked with 0.05% w\/v glutaraldehyde for 5\u2009min and quenched with 25\u2009mM Tris-HCl pH 7.5. For the first dataset, the reaction was vitrified without further purification as described for the pre-IC. For the second and third dataset, the reaction was purified after cross-linking through ALFA pull-down. Five cross-linked and quenched reactions were pooled and bound to 1.5\u2009\u00b5l ALFA Selector PE resin through the ALFA-tagged 2\u00d7MH-ARS1 DNA template for 1\u2009h at 24\u2009\u00b0C and 1,250\u2009rpm, washed once with 50\u2009\u00b5l buffer O and eluted for 30\u2009min at 24\u2009\u00b0C and 1,250\u2009rpm in 12\u2009\u00b5l buffer O\u2009+\u2009200\u2009\u00b5M ALFA peptide. Cryo-EM grids were prepared as described for the pre-IC.<\/p>\n Cryo-EM data collection for phospho-DH-3745<\/p>\n Three datasets of 31,794 (dataset 1), 32,200 (dataset 2) and 41,658 (dataset 3) movies were acquired on a 300-kV FEI Titan Krios G3i using a Gatan K2 Summit direct electron detector in counting mode and a BioQuantum energy filter with a slit width of 20\u2009eV at a nominal magnification of 130,000\u00d7 (1.08\u2009\u00c5\u2009px\u22121) using EPU v.3.2. Per hole, two shots were recorded with a 100-\u00b5m objective aperture inserted, a defocus range from \u22121.1 to \u22122.5\u2009\u00b5m and a total fluence of 49.1\u201350.4 electrons per \u00c52.<\/p>\n Cryo-EM image processing for phospho-DH-3745<\/p>\n Each dataset was preprocessed separately. First, cryo-EM movies were motion-corrected in RELION-4 using its own implementation of MotionCor281<\/a> and CTF estimation was done with Gctf78<\/a>. A Topaz picking network82<\/a> was iteratively trained on the first dataset using a selection threshold of \u22123, a scale factor of 8 and 30 expected particles per micrograph. Picked particles were extracted, 2\u00d7 binned (2.16\u2009\u00c5\u2009px\u22121) with a 360-px box size and cleaned up by reference-free 2D classification. Noise and low-quality averages were discarded. The remaining particles were used as input for the next round of Topaz training. Ultimately, particles were unbinned (448-px box size) and transferred to cryoSPARC80<\/a> to generate an initial volume, and subjected to non-uniform refinement with C2 symmetry applied. A total of 100,495 particles from dataset 1, 184,975 particles from dataset 2 and 239,120 particles from dataset 3 were then joined for downstream processing, yielding 524,590 particles. After another round of 2D classification, 359,025 particles underwent 2 rounds of CTF refinement (beam-tilt, anisotropic magnification, per-particle defocus and per-micrograph astigmatism) and Bayesian polishing in RELION83<\/a>, resulting in a 3.1-\u00c5 reconstruction of the consensus phospho-DH bound to Sld3 after non-uniform refinement in cryoSPARC with C2 symmetry applied.<\/p>\n To isolate Cdc45-bound phospho-DHs, particles were first C2-symmetry-expanded in cryoSPARC. An AlphaFold-Multimer84<\/a> prediction of an Sld3-CBD\u2013Cdc45 complex was aligned with the map of the phospho-DH by docking an atomic model of a CMG ring30<\/a> (PDB: 7QHS<\/a>) into one MCM ring and superimposing the prediction via Cdc45. A volume of the aligned Sld3-CBD\u2013Cdc45 prediction was generated at 10-\u00c5 resolution through the \u2018molmap\u2019 command in ChimeraX85<\/a> and used as input to prepare a soft mask in RELION. Using this mask, 718,050 C2-symmetry-expanded phospho-DH particles were subjected to 2 rounds of focused 3D classification without alignment and a T-value of 20 into 8 classes. A total of 72,693 particles exhibiting proteinaceous features inside this mask were selected and refined in RELION in C1 local searches restricted to 1.8\u00b0 while masking out Sld3-CBD\u2013Cdc45 signal from the opposite MCM hexamer. Subsequently, the masked out Sld3-CBD\u2013Cdc45 density was signal-subtracted in RELION. The Cdc45-bound DH was then imported into cryoSPARC and locally refined, yielding a final reconstruction at 3.7\u2009\u00c5.<\/p>\n In parallel, C2-symmetry-expanded Sld3-bound phospho-DHs were subjected to signal subtraction within cryoSPARC. Cdc45 and Sld3-CBD density associated with one of the two MCM hexamers was masked out and subtracted. A total of 718,050 particles of the remaining phospho-DH bound to a single Sld3-CBD\u2013Cdc45 complex were subjected to 2 rounds of 3D classification without alignment in cryoSPARC. Ten classes were chosen, with a class similarity of 0.25, and resolution was limited to 15\u2009\u00c5. A total of 96,027 particles were selected, from 3D classes displaying featured Cdc45 density, and locally refined using C1 symmetry in cryoSPARC to a resolution of 3.5\u2009\u00c5. This approach yielded a map of the Sld3\u2013Cdc45-bound phospho-DH, also featuring Sld7 density.<\/p>\n Cryo-EM sample preparation for sCMGE assembled with Sld2 and RPA<\/p>\n sCMGE complexes on double-roadblocked ARS1 DNA (168\u2009bp) were prepared essentially as described above (\u2018In vitro ATP\u2013dCMGE assembly\u2019). After dCMGE splitting, 20-\u00b5l assembly reactions were further purified on paramagnetic ALFA beads as described above (\u2018ALFA pull-down of pre-IC and dCMGE complexes\u2019). Cryo-EM grids were prepared as described for the pre-IC, with three applications of 4\u2009\u00b5l eluate per grid.<\/p>\n Cryo-EM data collection for sCMGE assembled with Sld2 and RPA<\/p>\n A total of 50,060 movies were collected at a nominal magnification of 130,000\u00d7 (0.95\u2009\u00c5\u2009px\u22121 physical pixel size) on a FEI Titan Krios G3i using a Falcon IV direct electron detector in counting mode and a Selectris energy filter with a slit width of 10\u2009eV using EPU v.3.2. Per hole, three shots were acquired with a defocus range from \u22122.0 to \u22122.9\u2009\u00b5m and a 100-\u00b5m objective aperture inserted. Movies were recorded with 31 frames and a total dose of 38.6 electrons per \u00c52 (Extended Data Table 2<\/a>).<\/p>\n Cryo-EM image processing of sCMGE assembled with Sld2 and RPA<\/p>\n A total of 50,060 EER movies were motion-corrected using RELION\u2019s own implementation of MotionCor281<\/a> and CTF-estimated with CTFFIND (v.4.1.13)86<\/a>. 1,940 particles were manually picked from 20 micrographs and used as input for Topaz training82<\/a>. Using Topaz, 4,827,716 particles were picked and extracted at 3.8\u2009\u00c5\u2009px\u22121 (4\u00d7 binning) and a 108-px box size, and cleaned up with multiple rounds of 2D classification in cryoSPARC (v.4.4.1)80<\/a>. A subset of clean sCMGE and MCM-DH classes were selected to generate initial 3D models, which were used in heterogenous refinement of a total of 3,801,543 sCMGE and MCM-DH particles. This yielded 1,125,095 sCMGE particles, which were unbinned with a 432-px box and homogeneously refined (with global CTF correction) to a final resolution of 2.7\u2009\u00c5.<\/p>\n Cryo-EM sample preparation for sCMGE assembled without Sld2 and RPA<\/p>\n sCMGE complexes were assembled as described above, with one notable exception: the omission of Sld2. After dCMGE splitting, 13 20-\u00b5l reactions were pooled and jointly purified on 60\u2009\u00b5l ALFA beads. Binding and washing steps were performed as described, and DNA-bound complexes were eluted in 50\u2009\u00b5l buffer O\u2009+\u2009200\u2009\u00b5M ALFA peptide. Lacey cryo-EM grids (400-mesh Cu; TAAB) were coated with graphene oxide by first hydrophilizing the grid surface with 4\u2009\u00b5l 300\u2009nM DDM and side-blotting, followed by two rounds of on-grid incubation with 4\u2009\u00b5l of a 20\u2009\u00b5g\u2009ml\u22121 graphene oxide suspension in 300\u2009nM DDM. Grids were washed with three 5-\u00b5l droplets of Milli-Q\u00a0H2O\u00a0from the backside, and blotted dry from the backside. Cryo-EM grids were vitrified as described before for the pre-IC, with two applications of 4\u2009\u00b5l eluate per grid.<\/p>\n Cryo-EM data collection for sCMGE assembled without Sld2 and RPA<\/p>\n A total of 70,337 movies were recorded on a FEI Titan Krios G3i at a nominal magnification of 130,000\u00d7 (0.95\u2009\u00c5\u2009px\u22121 physical pixel size) using a Falcon IV direct electron detector in counting mode and a Selectris energy filter with a slit width of 10\u2009eV using EPU v.3.2. Shots were acquired in a 0.7-\u00b5m spacing, with a 100-\u00b5m objective aperture inserted and a defocus range from \u22121.4 to \u22122.4\u2009\u00b5m. Per movie, 29 frames were recorded with a total fluence of 42.0 electrons per \u00c52 (Extended Data Table 2<\/a>).<\/p>\n Cryo-EM image processing for sCMGE assembled without Sld2 and RPA<\/p>\n A total of 70,337 EER movies were preprocessed using RELION\u2019s own implementation of MotionCor281<\/a> and imported into cryoSPARC (v.4.4.1)80<\/a> for CTF estimation using PatchCTF. A total of 4,579 particles were manually picked from 134 micrographs and used to train a Topaz model87<\/a> with 75 expected particles per micrograph. A total of 2,390,783 particles were extracted with 8\u00d7 binning at 7.6\u2009\u00c5 per px and a 70-px box size, and subsequently cleaned up with multiple rounds of reference-free 2D classification, removing well-averaging MCM-DHs as well as noise, ultimately yielding 162,691 CMG-like particles. Initial 3D references were generated with clean subsets of sCMGE- and dCMGE-like classes (orange and purple outlines, respectively; Extended Data Fig. 8c<\/a>) using cryoSPARCs ab-initio reconstruction. These volumes were subsequently used to separate sCMGEs from contaminating dCMGE-like particles through multiple rounds of heterogenous refinement (C1 symmetry; one sCMGE reference and two dCMGE references). A total of 72,370 cleaned-up sCMGE particles were then unbinned in RELION-5.0 with a box size of 512\u2009px, subjected to 2 rounds of Bayesian polishing and 3 rounds of CTF refinement (each round correcting separately: fourth-order aberrations, tilt and trefoil; anisotropic magnification; per-particle defocus and per-micrograph astigmatism) and 3D-refined using Blush88<\/a> to a nominal resolution of 3.4\u2009\u00c5.<\/p>\n Atomic model building and refinement<\/p>\n The structure of the pre-IC complex was built to a locally refined monomeric pre-IC cryo-EM map density-modified with EMReady89<\/a>. A single CMGE complex extracted from PDB 7PMK<\/a> (ref. 48<\/a>) was first docked into the cryo-EM density. After this initial placement, individual domains were then docked as rigid bodies into the cryo-EM density using UCSF Chimera90<\/a>. Because they matched the cryo-EM density closely, the structure predictions of Dpb11\u2013Mcm7, Dpb11\u2013GINS, Cdc45\u2013Sld3, Mcm4\u2013Sld3 and Mcm7\u2013Sld7 generated with AlphaFold\u2009337<\/a> were also used as starting\u00a0models for\u00a0building these interaction interfaces. Each chain or pair of chains was flexibly fit after generating self-restraints in Coot91<\/a> using density maps of varying blurring. Fragments mapping outside the visible density were deleted. The entire model was then manually adjusted with real-space refinement in Coot, using varying whole-molecule restraints depending on the local quality of the density. Automated real-space refinement was then performed in PHENIX (v.1.21)92<\/a> against the non-postprocessed map.<\/p>\n The pre-IC dimer complex structure was built to a C2-symmetric cryo-EM map density modified with EMReady. First, two refined pre-IC monomers were rigid-body-docked into the cryo-EM density map in ChimeraX. DNA chains from each monomer were trimmed at the overlapping region and were merged. Finally, the protein\u2013protein interface between the monomers was adjusted using real-space refinement in Coot. Automated real-space refinement was then performed in PHENIX (v.1.21)92<\/a> against the non-postprocessed map.<\/p>\n The DH\u2013Sld3-MBD and DH-3745 complex was built starting from a previously published MCM-DH structure bound to duplex DNA (PDB: 7P30<\/a>; ref. 14<\/a>), combined with the structure predictions of Mcm4\u2013Sld3, Sld7\u2013Mcm6 and Cdc45\u2013Sld3 produced using AlphaFold 2 (v.2.3.4)84<\/a>. Initial docking was performed in ChimeraX93<\/a>. The density fit of protein regions except zinc fingers was first adjusted using molecular-dynamics-based real-space refinement in Isolde94<\/a>. Next, the positions of all atoms were adjusted with flexible fitting and real-space refinement (sphere refinement) in Coot91<\/a>. Fragments mapping outside the visible density were truncated from the atomic coordinate file. Automated real-space refinement was performed against the non-postprocessed map.<\/p>\n The structure of sCMGE assembled on ARS1 DNA with RPA and Sld2 was modelled into a cryo-EM map density modified with EMReady, using PDB 7PMK<\/a> (ref. 48<\/a>) as an initial template. The starting model was rigid-body-docked in UCSF Chimera, followed by flexible fitting of individual chains in Coot using chain restraints. Where density permitted, the model was expanded manually or guided by AlphaFold 2 predictions. Because the resolution was insufficient for base identification, an arbitrary repetitive DNA sequence was modelled. To account for the bases that stretch between the modelled double-stranded and single-stranded DNA stretches and could not be built owing to poor density, we maintained the nucleotide numbering from PDB 6SKL<\/a> (ref. 95<\/a>). Final automated real-space refinement was performed using PHENIX (v.1.21) against the non-postprocessed map. For all structures, the quality of the resulting atomic models was evaluated with MolProbity96<\/a> (Extended Data Tables 1<\/a> and 2<\/a>). Structures in figures are displayed using EMReady-postprocessed maps.<\/p>\n Reporting summary<\/p>\n