Volume 54, Issue 5, Pages 879-886 (June 2014) Structural Basis for Hydroxymethylcytosine Recognition by the SRA Domain of UHRF2  Ting Zhou, Jun Xiong, Mingzhu Wang, Na Yang, Jiemin Wong, Bing Zhu, Rui-Ming Xu  Molecular Cell  Volume 54, Issue 5, Pages 879-886 (June 2014) DOI: 10.1016/j.molcel.2014.04.003 Copyright © 2014 Elsevier Inc. Terms and Conditions

Molecular Cell 2014 54, 879-886DOI: (10.1016/j.molcel.2014.04.003) Copyright © 2014 Elsevier Inc. Terms and Conditions

Figure 1 Overall Structure (A) Ribbon diagram showing UHRF2-SRA (green) bound to the 12 bp DNA containing a 5hmC base in a central CG site. The flipped-out 5hmC base is indicated, and secondary structural elements of UHRF2-SRA are labeled. (B) Superposition of a semitransparent surface representation of UHRF2-SRA showing the positively charged area involved in binding DNA. A narrow hydrophobic pocket accommodates the binding of the flipped-out 5hmC base, which is shown in a stick model (carbon, yellow; oxygen, red; nitrogen, blue; phosphorus, orange). See also Figure S1. Molecular Cell 2014 54, 879-886DOI: (10.1016/j.molcel.2014.04.003) Copyright © 2014 Elsevier Inc. Terms and Conditions

Figure 2 Protein-DNA Interaction (A) A schematic diagram detailing interactions between UHRF2-SRA and DNA. (B) 5hmC recognition by UHRF2 residues located on the β3-β4 loop (Ala492 to Glu498) and Tyr507, Thr508, and Gly509 located C-terminal to β4. 5hmC and the involved amino acids are shown in a stick model (carbon bonds are shown yellow and green for 5hmC and UHRF2-SRA residues, respectively). Water molecules are shown as red spheres, and hydrogen bonds are indicated with dashed magenta lines. (C) Val475 located at the tip of the α1-β2 loop fills the space vacated by the flipped-out 5hmC. Val45 is shown as a stick model superimposed with a dots representation, and DNA is displayed as a semitransparent surface superimposed onto a stick model. See also Figure S2. Molecular Cell 2014 54, 879-886DOI: (10.1016/j.molcel.2014.04.003) Copyright © 2014 Elsevier Inc. Terms and Conditions

Figure 3 Binding Preferences of the SRA Domains of UHRF2 and UHRF1 (A) Fluorescence polarization measurements of binding affinities between UHRF2, unmethylated (C/C), hemimethylated (M/C), hemihydroxymethylated (H/C), and fully hydroxymethylated (H/H) DNA (top panel). The bottom panel shows parallel measurements of the corresponding UHRF1 fragment. (B) Superposition of the 5hmC and 5mC binding pockets of UHRF2-SRA (green) and mUHRF1-SRA (silver; PDB ID: 2ZO1). The involved amino acid side chains are superimposed on the ribbon diagrams. 5hmC and 5mC are shown in a stick model with the carbon bonds colored yellow and magenta, respectively. (C) Tyr471 of mUHRF1-SRA generates a narrower binding pocket not optimally suited for 5hmC binding in comparison to Phe495 of UHRF2-SRA. (D) The smaller 5mC fits in the mUHRF1-SRA binding pocket well. See also Figures S2 and S3. Molecular Cell 2014 54, 879-886DOI: (10.1016/j.molcel.2014.04.003) Copyright © 2014 Elsevier Inc. Terms and Conditions

Figure 4 Binding of UHRF2 to Fully Hydroxymethylated DNA (A) EMSA showing the binding of UHRF2-SRA (aa 419–648) and a longer UHRF2 fragment to DNA probes containing 15 CG sites with unmethylated (C), fully methylated (mC), or fully hydroxymethylated cytosines (hmC). (B) Structure of UHRF2-SRA bound to a blunt-ended hemihydroxymethylated DNA with dual flipped-out 5hmC and the unmethylated cytosine in the opposite strand. The ribbon model of the UHRF2-SRA molecule bound to 5hmC is shown in green, and the UHRF2-SRA molecule bound to unmethylated cytosine is shown in cyan. The NKR loops of UHRF2-SRA are disordered, and the superimposed NKR loops of mUHRF1-SRA, shown in magenta and blue, sterically clash with each other. See also Figures S3 and S4. Molecular Cell 2014 54, 879-886DOI: (10.1016/j.molcel.2014.04.003) Copyright © 2014 Elsevier Inc. Terms and Conditions