1 The SRA domain of UHRF1 flips 5-methylcytosine out of the DNA helix

doi: 10.1038/nature07280 www.nature.com/nature 1 2 Figure S1. Mapping the SRA domain within mouse UHRF1 a, Mouse UHRF1 (mUHRF1) is a 782-residue protein containing four recognizable domains including a ubiquitin-like domain, a plant homeodomain (PHD) that may be involved in histone H3 tail binding 1, 2, a SET and RING associated (SRA) domain that preferentially binds to hemimethylated CpG sites 3, and a C-terminal Really Interesting New Gene (RING) domain may confer E3 ubiquitin ligase activity 1. b, Proteolytic digestion, mass spectrometry, and deletion analyses identified the SRA domain boundaries of mUHRF1 as residues 419-628. Left panel: SDS-PAGE gel of the purified recombinant mUHRF1 full length and its trypsin digestion products. Purified hexahistidine-SUMO-tagged mUHRF1 full length protein (1.7 µg) in 20 mM Hepes 7.0, 400 mM NaCl, 5 % glycerol, and 0.1 % 2-mercaptoethanol was treated with 0, 0.5, 5, 50 ng of trypsin for 30 min and separated on a 13 % SDS gel. Mass spectrometry determined molecular masses of individual fragments. Each fragment was constructed into a hexahistidine-SUMO tagged fusion protein and expressed in Escherichia coli. We also varied the starting and ending amino acids of fragments to reach maximum expression and solubility. Right panel: SDS-PAGE gel of the purified recombinant SRA domain (between the molecular weight markers of 20 and 26.6 kDa) used for crystallization. References: 1. Citterio, E. et al. Np95 is a histone-binding protein endowed with ubiquitin ligas