GoKawiilProtein expression and purification
HpaII methyltransferase (MH), ORC, Cdc6, Mcm2–7–Cdt1, DDK, CDK, (yeast-expressed) Sld3/7, Cdc45, GINS, (yeast-expressed) Pol ε, Mcm10, RPA, topoisomerase I (TopoI), Pol α and Rad53 were expressed and purified as previously described24,30,40,46,66,67,68,69. 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.
Cell lines
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.
Cloning, expression and purification of Twin-Strep-tagged Sld3/7
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 and transformed into electrocompetent EMBacY cells (Geneva Biotech). Cells were screened by blue–white selection for successful bacmid integration and selected colonies were grown overnight at 37 °C. Cells were collected by centrifugation and bacmids were purified by isopropanol precipitation. A total of 45 µg bacmid DNA was mixed thoroughly with 13.5 µl FuGENE HD Transfection Reagent (Promega) and 450 µl Sf-900 III SFM medium and incubated for 30 min at room temperature. Two hundred microlitres of transfection mix was added dropwise to 2 ml Sf21 insect cells seeded at 0.5 million cells per ml in a six-well plate. Plates were incubated for 3–5 days at 27 °C 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 ml Sf21 insect cells at 0.5 million cells per ml were inoculated with 4 ml P0 baculovirus in suspension and cultured at 27 °C shaking at 120 rpm. After viability had dropped below 90%, cells were pelleted by centrifugation at 380g for 15 min at 4 °C, and supernatant containing the amplified P1 virus was sterile-filtered (0.22 µm pore size) and stored at 4 °C. One litre of Sf21 insect cells were seeded at one million cells per ml in Sf-900 III SFM medium and infected with 0.5 % v/v P1 virus. Cells were collected 48 h after baculovirus-induced cell-cycle arrest by centrifugation for 15 min at 180g at 4° C, and pellets were flash-frozen in liquid nitrogen and stored at −80 °C.
Cells were thawed and resuspended in 50 ml buffer A (25 mM HEPES-KOH pH 7.5, 500 mM KCl, 10% v/v glycerol, 0.02% w/v NP-40, 1 mM EDTA and 1 mM DTT) supplemented with one cOmplete EDTA-free protease inhibitor tablet (Merck) and 0.7 mM phenylmethylsulfonyl fluoride (PMSF), then lysed by sonication on ice for 2 min (1-s pulse-on, 4-s pulse-off). The lysate was clarified by ultracentrifugation for 1 h at 45,000 rpm in a Ti45 rotor (Beckman) at 4 °C, and the supernatant was mixed with 2.4 ml Bio-Lock (IBA) reagent and applied onto 1 ml pre-equilibrated Strep-Tactin XT Superflow HighCapacity resin in a gravity column. The resin was washed with 100 ml buffer A and 10 mL buffer A supplemented with 2 mM ATP and 10 mM MgCl 2 . Protein was eluted with 10 ml 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 mg ml−1, aliquoted and flash-frozen in liquid nitrogen.
Cloning, expression and purification of Dpb11
Codon-optimized S. cerevisiae Dpb11 followed by a 3C protease cleavage site and a C-terminal 3×Flag 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 l Sf21 insect cells at one million cells per ml were infected with 0.5% v/v P1 virus and collected 48 h after cell-cycle arrest.
The cell pellet was resuspended in 50 mL buffer A supplemented with one cOmplete EDTA-free protease inhibitor tablet (Merck) and 0.7 mM PMSF, lysed by sonication on ice for 2 min (1-s pulse-on, 4-s pulse-off) and ultracentrifuged at 45,000 rpm at 4 °C for 1 h. The soluble phase was mixed with 2.4 ml Bio-Lock Reagent and passed through 1 ml pre-equilibrated anti-Flag M2 Affinity Gel (Sigma) in a gravity column. The column was washed with 150 ml buffer A and 10 ml buffer A supplemented with 2 mM ATP and 10 mM MgCl 2 . To elute bead-bound protein, the beads were resuspended in 5 ml buffer A supplemented with 0.5 mg ml−1 3×Flag peptide and incubated for 5 min, after which the flow-through was collected. The eluate was diluted to 150 mM KCl and loaded onto a 1 ml Mono S 5/50 column (Cytiva) equilibrated in buffer B (25 mM HEPES-KOH pH 7.5, 150 mM KCl, 10% v/v glycerol, 0.02% w/v NP-40, 1 mM EDTA and 1 mM DTT). After washing the column with 10 ml buffer B, Dpb11 was eluted with a linear gradient of 150–1,000 mM KCl in buffer B over 20 column volumes. Fractions containing pure Dpb11 were pooled and dialysed against buffer C (25 mM HEPES-KOH pH 7.5, 300 mM KOAc, 10% v/v glycerol, 0.02% NP-40, 1 mM EDTA and 1 mM DTT) at 4 °C overnight while stirring. Subsequently, Dpb11 was concentrated to approximately 0.5 mg ml−2, aliquoted and flash-frozen in liquid nitrogen.
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