Methylated ubiquitin

Yeast cells carrying Cdc16-TAP and lacking Cdh1 were collected at OD₆₀₀ = 1, flash frozen, and lysed using beat beating in lysis buffer. APC/C was immunopurified on IgG beads and activated by in vitro-translated Cdc20 or Cdh1. Cdc20 and Cdh1 used in Fig. 5e–g were expressed in insect cells using the baculovirus system and purified using Strep-tag-purification methods. For APC/C substrates, Pds1, Hsl1 fragment (aa 667–872), and Clb5 were C-terminally ZZ-tagged, cloned into plasmids under control of the T7 promoter, and translated in vitro using ³⁵S-Methionine. Substrates were purified using IgG-coupled magnetic beads and cleaved with TEV protease (Thermo Fisher #12575015). E1 and E2 (Ubc4) were expressed in E. coli and purified^{60 (link),61 (link)}. E2 charging was performed in a reaction containing 0.2 mg/ml Uba1, 2 mg/ml Ubc4, 2 mg/ml methylated ubiquitin (Boston Biochem #U-501) and 1 mM ATP at 37 °C for 30 min. The ubiquitylation reaction was initiated by mixing activated APC/C, E2-ubiquitin conjugates, purified substrate, and buffer or indicated concentrations of single-stranded DNA at 25 °C. Reactions were terminated by addition of 2X SDS sample loading dye and separated by SDS–PAGE.

APC/C ubiquitylation assays were performed as described previously^{24 (link)}. In short, APC/C was purified from yeast as described above using magnetic IgG beads and activated with a purified Cdh1 or Cdc20 activator. For all APC/C ubiquitylation assays, substrates were produced by in vitro translation with ³⁵S-Methionine (see Supplementary Table 5 for plasmids). Substrates were purified using magnetic IgG beads. E1 and E2 (Ubc4) were expressed in E. coli and purified as described previously^{25 (link),42 (link)}. E2 was charged at 37 °C for 30 min (reaction contained 0.2 mg ml⁻¹ Uba1, 2 mg ml⁻¹ Ubc4, 2 mg ml⁻¹ methylated ubiquitin from Boston Biochem #Y-501, and 1 mM ATP). Charged E2 was added to a reaction containing activated APC/C and substrate. Reaction products were analyzed by SDS-PAGE and Phosphorimaging on a Typhoon 9400 Imager.

E3 ligase assays were performed in the presence of reconstituted PRC1 and nucleosomes, with the addition of UBE1 (Boston Biochem), UbcH5c (Boston Biochem), methylated ubiquitin (Boston Biochem) and ATP (Life Technologies). Concentrations of E1, E2 and ubiquitin were pre-optimised such that reactions were pseudo-first order with respect to PRC1. Ubiquitylation reactions were carried out in 50 mM Tris (pH 7.5), 2.5 mM MgCl₂ and 0.5 mM DTT. E1, E2, ubiquitin and ATP were pre-incubated for 20 min at 37°C, followed by addition of PRC1 and nucleosomes (0.35 µM final concentration), and then incubated at 37°C for 1 hr. Reactions were quenched with 5 mM EDTA and SDS-PAGE loading buffer (2% SDS, 100 mM Tris-HCl (pH 6.8), 100 mM DTT, 10% glycerol, 1 mg/mL bromophenol blue), boiled for 5 min, and loaded onto SDS-PAGE gels for western blot analysis. Western blots were probed with antibodies specific for histone H2AK119ub1 (Farcas et al., 2012 (link)) or histone H2A (Millipore 07–146), followed by incubation with LiCOR IRDye secondary antibodies (800CW goat anti-rabbit and goat anti-mouse). Westerns were imaged using the LiCOR Odyssey Fc imaging system, and the fraction of H2AK119ub1 relative to total H2A (non-ubiquitylated H2A plus H2AK119ub1) was quantified. Data were visualised and dose-response curves fitted using GraphPad Prism 6.0.