Structure of the pre-initiation complex explains CMGE biogenesis

Protein 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 described 24 , 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 assembly 70 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. …