GoKawiilCompounds
dBET6 (HY-112588), dBRD9 (HY-117690), MLN7243 (HY-100487), MLN4924 (HY-70062), MG132 (HY-13259), bafilomycin A 1 (HY-100558), and 5K Scaffold Library (HY-L902) were obtained from MedChemExpress.
Plasmids
The following plasmids were used in this study: Artichoke (Addgene #73320) and Cilantro (Addgene #74450) for flow-based reporter degradation assays, reporter CRISPR screens and co-immunoprecipitation; sgBFP (U6.sgRNA.SFFV.tBFP) for validation of DCAF11-knockout phenotypes; pNTM2 (CMV) for co-immunoprecipitation; pAC8-derived plasmids for protein purification. All proteins are derived from human origin sequences: full-length DCAF11, DDX18 (residues 171–625), DDB1(ΔBPB) (residues 1–395 and 706–1140 with a GNGNSG linker) and full-length DDA1. Unique amino acid tags: Flag (for DCAF11, DDX18), Flag–GFP (for DDX18), StrepII–Avi (for DCAF11 and DDX18) and His (for DDB1(ΔBPB) and DDA1) were designed to the N-terminal ends of the constructs, and then subcloned into pAC-derived expression vectors (pAC8RedNK)45.
Antibodies
The following antibodies were used: Flag (CST 14793S), GFP (CST 2555S), β-actin (CST 3700S), tubulin (Sigma T9026), BRD4 (Bethyl A301-985A-T), DDX18 (GeneTex GTX103392), BRD9 (Bethyl, A303-781A-T), SMARCA2 (Bethyl A301-015A-T), LIMK2 (CST 3845 T), WEE1 (CST 4936S), CDK7 (Proteintech 27027-1-AP), cyclin H (Proteintech 67065-1-lg), MNAT1 (Proteintech 11719-1-AP), DCAF11 (Novus Biologicals NBP2-92244), IRDye 800CW Goat anti-Rabbit IgG Secondary Antibody (LI-COR 926-32211) and IRDye 680LT Goat anti-Rabbit IgG Secondary Antibody (LI-COR 925-68021).
Protein expression and purification
The recombinant proteins from the constructs in pAC-derived vectors were expressed in Trichoplusia ni High Five insect cells (Gibco, 85502) using the baculovirus expression system. In brief, expression plasmids were transfected into Spodoptera frugiperda (Sf9) cells (Expression Systems, 94-001 F) at a density of 0.9 × 106 cells per ml grown in ESF 921 medium (Expression Systems) to generate baculovirus, and this was followed by 2 rounds of infection in Sf9 cells to increase viral titre. For recombinant protein expression, High Five cells grown in SF-4 baculo express insect medium (BioConcept) at a density of 2.0 × 106 cells per ml were infected with baculovirus at 1.5% v/v ratio. After 42 h of expression at 27 °C, High Five cells were collected by centrifugation for 15 min at 1,500 rpm. For purification of StrepII or Flag-tagged proteins, pelleted cells were resuspended in lysis buffer containing 50 mM Tris (hydroxymethyl) aminomethane hydrochloride (Tris-HCl) pH 8.0, 200 mM NaCl, 1 mM Tris (2-carboxyethyl) phosphine (TCEP), and protease inhibitors, and the cell pellets were lysed by sonication. After ultracentrifugation (1 h, 40,000 rpm, 4 °C), the soluble fraction was passed over the appropriate affinity resin of Strep-Tactin XT Superflow (IBA 2-4010-025) or Anti-DYKDDDDK G1 Affinity Resin (Genscript L00432), eluted with wash buffer (50 mM Tris-HCl pH 8.0, 200 mM NaCl, 1 mM TCEP) supplemented with 50 mM Biotin (IBA 2-1016-005) or 0.15 mg ml−1 Flag peptide (custom synthesis), respectively. The affinity-purified proteins were then applied to an ion exchange column (POROS 50HQ, Thermo Scientific 1255911) and eluted in 50 mM Tris-HCl pH 8.5 and 2 mM TCEP by a linear salt gradient (from 50 mM to 1000 mM NaCl). All proteins were then subjected to size-exclusion chromatography on a Superdex 200 Increase 10/300 (Cytiva 28990944) in 25 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) pH 7.4 or pH 8.0, 150 mM NaCl and 1 mM TCEP. For structural biology studies, the non-concentrated peak fraction was used, whereas for biochemistry studies, protein within the peak was pooled, concentrated, and flash frozen in liquid nitrogen and stored at −80 °C.
Biotinylation
Purified StrepII–Avi-tagged DCAF11 or DDX18 were biotinylated in vitro by incubation with final concentrations of 2.5 μM BirA enzyme (prepared in-house) and 0.2 mM biotin in 50 mM HEPES, pH 7.4, 200 mM NaCl, 10 mM MgCl 2 , 1 mM TCEP and 20 mM ATP. The reaction was incubated for 1 h at room temperature and stored overnight at 4 °C. Biotinylated proteins were purified by size-exclusion chromatography and flash frozen in liquid nitrogen and stored at −80 °C.
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