Seth Wichmann posted an update 3 weeks, 1 day ago
After with water then released in the G2/M arrest in YP-Gal at 37 . two glucose was added to the cultures in the indicated instances unless stated otherwise.Pre-RC assayThe pre-RC assay was performed as described with minor SSR240612 In Vivo modifications (Evrin et al., 2009). Right here, a one-step reaction was used. 40 nM ORC, 80 nM (wt or mutant) Cdc6, 40 nM Cdt1, 40 nM MCM2-7 in buffer A (50 mM HEPES-KOH pH 7.five, one hundred mM KGlu, ten mM MgAc, 50 M ZnAc, 3 mM ATP, five mM DTT, 0.1 Triton X-100, and 5 glycerol) were added to six nM linear pUC19-ARS1 DNA coupled to magnetic beads for 15 min at 24 . Beads had been washed two occasions with buffer A containing 300 mM KGlu plus 1 mM EDTA, or two?short washes with buffer A-1 (50 mM HEPES-KOH pH 7.five, 1 mM EDTA, 300 mM KAc, ten glycerol, 0.1 Triton X-100, and five mM DTT), or 3?with buffer B (50 mM HEPES-KOH pH 7.five, 1 mM EDTA, 500 mM NaCl, 10 glycerol, 0.1 Triton X-100, and five mM DTT) prior to digestion with 1 U of DNase I in buffer A plus 5 mM CaCl2 for 2 min at 24 . The samples were separated by SDS-PAGE and analyzed by silver staining.Expression and purification of proteinsORC was expressed by utilizing baculovirus-infected cells and purified as described (Klemm et al., 1997). Cdc6 (WT and mutants) and Cdt1 have been expressed in bacteria and purified as described (Speck et al., 2005; Evrin et al., 2009). Mcm2-7 were expressed in Saccharomyces cerevisiae and purified as described (Evrin et al., 2009).Chang et al. eLife 2015;4:e05795. DOI: 10.7554/eLife.11 ofShort reportGenes and chromosomesIn vitro assembly and purification with the OCCM and Mcm2-7 doublehexamer complexes for single particle EMThe pre-RC had been assembled in a one-step reaction: 40 nM ORC, 80 nM Cdc6, 40 nM Cdt1, and 40 nM MCM2? in buffer A (50 mM HEPES OH pH 7.5, one hundred mM KGlu, 10 mM MgAc, 50 mM ZnAc, 3 mM ATP, 5 mM DTT, 0.1 Triton X-100, and 5 glycerol) have been added to 6 nM pUC19-ARS1 plasmid beads at 24 (Evrin et al., 2009). After 10 min (for pre-RC intermediate analysis) or 40 min (for MCM2-7 double-hexamer evaluation), the beads have been washed three occasions with buffer A (pre-RC intermediate) or B (MCM2-7 double-hexamer) and three instances with buffer C (50 mM HEPES-KOH [pH 7.5], one hundred mM potassium acetate, 5 mM magnesium acetate, 5 mM CaCl2) and eluted with 1 U DNase I in five l buffer C.ATPase assayThe ATPase assay was performed as described (Speck and Stillman, 2007; Fernandez-Cid et al., 2013). Error bars represent the normal deviation from at the least 3 independent experiments.EM grids preparationThe concentration of pre-RC intermediates and loaded dhMCM was low for cryo-EM. To obtain extra particles in every single image, we added a thin continuous carbon on best of your commercially available lacey carbon grids (SPI #3830C-MB).