SUPPLEMENTARY INFORMATION

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1 Supplementary Results DNA binding property of the SRA domain was examined by an electrophoresis mobility shift assay (EMSA) using synthesized 12-bp oligonucleotide duplexes containing unmodified, hemi-methylated, or fully methylated CpG (Supplementary Fig. 1a). The SRA domain preferentially bound to hemi-methylated DNA over unmodified or fully methylated DNA (Supplementary Fig. 1b). Co-crystallization was performed with the hemi-methyl CpG-1 and CpG-2 oligonucleotides, and we determined three crystal structures of the complexes: the SRA:hemi-methyl CpG-1 complex crystallized in two different crystal forms, monoclinic C2 and tetragonal P ; the SRA:hemi-methyl CpG-2 DNA complex crystallized in space group P These complex structures are essentially identical in both protein and DNA portions (Supplementary Fig. 9), suggesting that conformational changes of the SRA domain upon DNA binding are not caused by crystal packing effects. In the monoclinic form, the dual conformations were locally observed in one of the asymmetric molecules; the main chain of the region Ala468-Gly470 in chain A and the G 8 phosphate backbone in chain D. We classified one of the dual conformations in theses regions, which was also observed in another asymmetric molecule with full occupancy, as conformation I, and the other as conformation II. All structural figures are produced based on the structure with conformation I. These regions, Ala468-Gly470 and the G 8 phosphate backbone, in the tetragonal forms adopt conformation II. However, a common mode of base recognition by the SRA is observed in the both complexes with the hemi-methyl CpG-1 and CpG-2 DNAs. (Fig. 2b, Supplementary Fig. 10 and Supplementary Tables 2, 3). 1

2 Supplementary Figure 1 Specific binding of SRA to the hemi-methylated DNA. a, Oligonucleotide sequences used in the crystallization and/or mobility shift assays: C, cytosine; G, guanine; A, adenine; T, thymine; M, 5-methyl cytosine. b, An electrophoretic mobility shift DNA-binding assay using the 12-bp oligonucleotides shown in (a). Poly (di-dc) was used as a nonspecific competitor (see Supplementary Methods). The SRA:hemi-methyl CpG-1 and SRA:hemi-methyl CpG-2 complexes each showed a clear shift band, whereas no complex with full-methylated or unmethylated DNA was detected. Supplementary Figure 1 2

3 Supplementary Figure 2 Folding topology diagram of the SRA domain in the hemi-methylated DNA-bound state. α-helices and β-strands are shown by orange cylinders and blue arrows, respectively. Supplementary Figure 2 3

4 Supplementary Figure 3 Multiple sequence alignment of eukaryotic SRA domains. Sequence numbering is based on mouse UHRF1. Secondary structural elements of the SRA are indicated above the sequences, and disordered regions are shown in red broken lines. Amino acid residues are highlighted in a color code corresponding to conservation level: fully conserved, orange; conservative substitutions, light green. Supplementary Figure 3 4

5 Supplementary Figure 4 Electrostatic potential of the SRA domain. Surface colors represent potential from -15K B T -1 (red) to 15 K B T -1 (blue). a, Electrostatic potential of the unliganded SRA domain. Orange circle indicates the 5-methyl cytosine binding pocket. The SRA molecules are in the same orientations as in Fig. 1(a). b, Electrostatic potential of the SRA domain in complex with hemi-methylated DNA. The hemi-methylated DNA is shown in green. The complex molecules are shown in the same orientations as in Fig. 2(a). Supplementary Figure 4 5

6 Supplementary Figure 5 The SRA domain undergoes conformational changes upon DNA-binding. a, The structure of the unliganded SRA domain (beige) is superimposed on to the structure of the SRA domain in the DNA bound form (pale purple). The root mean square deviation (r.m.s.d) of the positions of 190 Cα atoms is 1.1 Å. The loop L3 in the DNA bound form is shown in light blue. The N-tail and finger loop in the DNA bound form are colored in orange. b, Close-up view of the DNA recognition by the N-tail (orange stick model) on the minor groove side. The electron density displayed is an Fo - Fc map calculated in the absence of the N-tail at 3.0 σ contour level. The methylated and unmethylated DNA strands are shown in light green and pale green, respectively. The 5-methyl cytosine, 5mC 7 is shown in magenta. c, Close-up view of the DNA recognition by the finger loop (orange stick model) on the major groove side. The electron density displayed is an Fo - Fc map calculated in the absence of the finger loop at 3.0 σ contour level. d, A structural change in the loop L3 is involved in recognition of 5mC 7. Upon DNA binding, the loop L3 is shifted ~2.9 Å closer to 5mC 7, as indicated by blue arrows. Supplementary Figure 5 6

7 Supplementary Figure 6 Recognition of the 5mC 7 methyl group by the SRA residues. Stereo view of the 5mC 7 binding pocket in the SRA domain. Red and black broken lines show possible hydrogen bonds within 4.0 Å and hydrophobic interactions between the methyl group of 5mC 7 and SRA, respectively. Supplementary Figure 6 7

Supplementary Figure 7 A conjectural model of the catalytic domain of Dnmt1 bound to DNA containing. Hemi-methylated CpG site The model is overlaid on the structure of the SRA-DNA complex.

8 Supplementary Figure 7 A conjectural model of the catalytic domain of Dnmt1 bound to DNA containing. Hemi-methylated CpG site The model is overlaid on the structure of the SRA-DNA complex. The 5-methyl cytosine recognized by the SRA domain is shown in magenta, and the target cytosine for Dnmt1 in blue. Close-up views of the minor and major groove sides are shown in left and right boxes, respectively. Supplementary Figure 7 8

Supplementary Figure 8 2 Fo - Fc electron density map of the SRA domain:hemi-methyl CpG-1 complex at 1.6 Å resolution. The density was contoured at 1.0 σ.

9 Supplementary Figure 8 2 Fo - Fc electron density map of the SRA domain:hemi-methyl CpG-1 complex at 1.6 Å resolution. The density was contoured at 1.0 σ. Blue and green stick models represent the SRA domain and the DNA molecule, respectively. Water molecules are shown as red spheres. The side chains of Arg496 and Val451 invade the vacant space after the base flipping of 5mC 7. Supplementary Figure 8 9

Supplementary Figure 9 Structure comparison of the SAR-DNA complexes. a, the SRA:CpG-1 complex in space group C2 (green) vs. the SRA:CpG-1 complex in P4 1 2 1 2 (red).

10 Supplementary Figure 9 Structure comparison of the SAR-DNA complexes. a, the SRA:CpG-1 complex in space group C2 (green) vs. the SRA:CpG-1 complex in P (red). b, the SRA:CpG-1 compelx in space group C2 (green) vs. the SRA:CpG-2 complex in space group P (blue). c, the SRA:CpG-1 complex in space group P (red) vs. the SRA:CpG-2 complex in space group P (blue). The r.m.s.d values calculated in the positions of Cα atoms between the SRA molecules are shown below each image. Supplementary Figure

11 Supplementary Figure 10 Schematic diagram of the protein-dna contacts in the SRA: hemi-methyl CpG-2 complex. Bases in the hemi-methylated 5mCpG/CpG site, G 7, G 8, and C 8 are colored in green, and 5mC 7 is colored in pink. Amino acid residues involved in DNA recognition are indicated, and those located in the N-tail and finger loop are enclosed by an orange box. Hydrogen bonds between DNA and the SRA are shown in red lines: the main chain contacts, dotted ones; the side chain contacts, solid ones. Blue lines show van der Waals contacts. W represents water molecules. Supplementary Figure

12 Supplementary Table 1 Crystallographic data and data collection statistics Crystal unliganded SRA-CpG1 SRA-CpG1 SRA-CpG2 Source PF-BL5 Detector ADSC Q315 Wavelength (Å) Space group P1 C2 P P Cell dimension a (Å) b (Å) c (Å) α (º) β (º) γ (º) Resolution range (Å) Total Observations 362, ,540 63, ,336 Unique reflections 96,644 77,191 10,753 11,160 R merge (%) 1, (37.2) 6.2 (32.9) 9.3 (26.3) 7.9 (17.1) Completeness (%) (77.3) 99.1 (91.9) 94.8 (79.1) 99.6 (100) I /σ<i> Redundancy (2.8) 4.0 (3.2) 5.9 (2.7) 9.8 (9.2) Refinement statistics Resolution range (Å) R work (%) 2, (21.4) 15.5 (18.2) 21.9 (27.6) 21.4 (25.2) R free (%) 2, (24.0) 18.5 (22.5) 27.5 (38.2) 27.5 (38.2) R. M. S. Deviation Bond angle (º) Bond length (Å) Number of atoms Protein 5,957 3,429 1,647 1,641 DNA Water Ligand Mean B values (Å 2 ) Main chain Side chain DNA Water R merge = Σ h Σ i I(h) i - <I(h)> / Σ h Σ i I(h) i,where I(h) is the intensity of reflection h, Σ h is the sum of all measured reflections and Σ i is the sum of i measurements of reflection. 2 R work and R free = (Σ hkl Fo - Fc ) / Σ hkl Fo, where the free reflections (5% of the total used) were held aside for R free throughout refinement. 3 Numbers in parentheses are the values for the highest resolution shell of each data set. 12

13 Supplementary Table 2 List of hydrogen bonds observed between the SRA domain and the bases of hemi-methyl CpG-1 in space group C2. Chain A Chain B Base Atom Residue/Base Atom distance (Å) distance (Å) 5mC 7 O2 Ala468 N (Gly469 N 2.95) # - N3 Asp474 Oδ N4 Asp474 Oδ Thr484 O m 5mC 7 O1P (CH-O bond) Tyr483 Oη (CH-O bond) Thr484 O (CH-O bond) Gly485 O (CH-O bond) G 7 ' N2 Ala407 O Val409 O O6 Agr496 Nε N7 Arg496 Nη G 8 N2 Ala407 O 2.85* 2.85 O6 Water O 2.80* 2.83 Arg496 N (Water mediate) Thr497 N (Water mediate) N7 Water O 2.79* 2.79 Thr497 Oγ (Water mediate) C 8 ' N4 Asn494 O * The average distances from the SRA residue to the dual conformers of G8. # The hydrogen bond in conformation II 13

14 Supplementary Table 3 List of hydrogen bonds observed between the SRA domain and the bases of hemi-methyl CpG-2. Base Atom Residue Atom distance (Å) 5mC 7 O2 Ala468 N 2.83 Gly469 N 3.14 N3 Asp474 Oδ N4 Asp474 Oδ Thr484 O m 5mC 7 O1P 4.06 (CH-O bond) Tyr483 Oη 3.40 (CH-O bond) Thr484 O 3.64 (CH-O bond) Gly485 O 4.25 (CH-O bond) G 7 ' N2 Ala407 O 3.30 Val409 O 2.98 O6 Agr496 Nε 2.69 N7 Arg496 Nη 2.71 G 8 N2 Ala407 O 2.88 C 8 ' N4 Asn494 O

SUPPLEMENTARY INFORMATION

SUPPLEMENTARY INFORMATION doi:10.1038/nature11524 Supplementary discussion Functional analysis of the sugar porter family (SP) signature motifs. As seen in Fig. 5c, single point mutation of the conserved