Purification and Binding Assays of UHRF1 Domains

His-MBP-tagged UHRF1 SRA-RING (amino acids 405–793) was produced in E. coli as described above. GST-tagged UHRF1 TTD-PHD (amino acids 123–366) and BPTF PHD-Bromo (gift from Dr. Alex Ruthenburg [Ruthenburg et al., 2011 (link)]) were produced as previously described (Rothbart et al., 2013 (link)). Proteins (each at 1 μM) were incubated overnight at 4°C with MBP magnetic beads (NEB, Ipswich, MA) in binding buffer containing 50 mM Tris-HCl, pH 8.0, 100 mM NaCl, 0.1% NP-40, 0.5% BSA, and, where indicated, 25 μM DNA oligonucleotides or histone peptides. Pulldown experiments with the SRA-RING DNA^mut were performed with 5 μM DNA. Samples were washed extensively with binding buffer, eluted in SDS sample buffer, resolved by SDS-PAGE, transferred to PVDF membrane (Thermo), and probed with GST antibody (Sigma #G7781, 1:2,000). Pull-down assays were performed in triplicate.