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. 2011;6(6):e21306.
doi: 10.1371/journal.pone.0021306. Epub 2011 Jun 22.

Recognition of 5-hydroxymethylcytosine by the Uhrf1 SRA domain

Carina Frauer  1 , Thomas Hoffmann, Sebastian Bultmann, Valentina Casa, M Cristina Cardoso, Iris Antes, Heinrich Leonhardt
Affiliations

Recognition of 5-hydroxymethylcytosine by the Uhrf1 SRA domain

Carina Frauer et al. PLoS One. 2011.

Abstract

Recent discovery of 5-hydroxymethylcytosine (5hmC) in genomic DNA raises the question how this sixth base is recognized by cellular proteins. In contrast to the methyl-CpG binding domain (MBD) of MeCP2, we found that the SRA domain of Uhrf1, an essential factor in DNA maintenance methylation, binds 5hmC and 5-methylcytosine containing substrates with similar affinity. Based on the co-crystal structure, we performed molecular dynamics simulations of the SRA:DNA complex with the flipped cytosine base carrying either of these epigenetic modifications. Our data indicate that the SRA binding pocket can accommodate 5hmC and stabilizes the flipped base by hydrogen bond formation with the hydroxyl group.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. DNA binding specificity of 5-methylcytosine binding proteins.
(A+B) Relative DNA/protein ratios of Uhrf1, its SRA domain (SRAUhrf1) and the MBD of MeCP2 (MBDMeCP2) with two differentially labeled DNA substrates in direct competition. (A) Binding to DNA substrates containing a hemimethylated or hemihydroxymethylated CpG site (HMB versus HhMB, respectively). (B) Binding to DNA substrates containing a fully methylated or fully hydroxymethylated CpG site (FMB versus FhMB, respectively). Results are shown as means of three independent experiments with standard deviation error bars. Note that MBDMeCP2 preferentially binds to FMB, whereas the Uhrf1 constructs do not discriminate between FMB and FhMB. (C) Electrophoretic mobility shift assays were performed with Uhrf1 or MBDMeCP2 and equimolar amounts of FMB (red) and FhMB (green) in competition. The overlay of the two substrate channels reveals simultaneous shifting of both DNA substrates with Uhrf1, whereas with MBDMeCP2 the FMB substrate shifts at a lower protein concentration than the FhMB substrate, confirming differential binding.
Figure 2
Figure 2. Structure of the Uhrf1 SRA domain in complex with hemimethylated and hemihydroxymethylated DNA.
(A) Experimental structure of the Uhrf1 SRA domain in complex with hemimethylated DNA (PDB-ID:3fde, [14]). The protein is shown in cartoon and the DNA in licorice representation. The 5mC nucleotide is highlighted in green. Note that the 5mC residue is flipped out of the DNA double helix. (B+C) Models of the SRA binding pocket with bound 5mC (B) and 5hmC (C) serving as starting points for the molecular dynamics simulations. The location of the hydroxyl group in the 5hmC complex is highlighted by the white arrow. The view is from the top of the binding site (DNA backbone) and rotated by 90 degrees compared to (A).
Figure 3
Figure 3. Molecular dynamics simulations of the SRA domain in complex with 5mC and 5hmC containing DNA.
(A+B) Three and two-dimensional schematic drawings summarizing the hydrogen bond networks between the nucleotides, the SRA binding pocket, and a conserved water molecule during the simulations. The numbers in (B) correspond to the numbering in (C+D). (C+D) Hydrogen bond occurrences during the molecular dynamics simulations of the SRA domain in complex with either 5mC (C) or 5hmC containing DNA (D). Each vertical line represents a single observed hydrogen bond. The hydrogen bond between 5hmC and the conserved water is highlighted in red.
Figure 4
Figure 4. Hydrogen bond networks stabilizing 5mC and 5hmC within the SRA binding pocket.
(A) SRA complex with DNA containing 5mC. (B+C) SRA complex with DNA containing 5hmC. In the 5hmC complex, the water molecule stably interacts with the hydroxyl group of the nucleotide, but two alternative conformations of the SRA binding pocket exist depending on the ion concentration. In the absence of salt, binding involves an interaction of the S486 residue with the phosphate group of the flipped nucleotide (B), whereas in the presence of 0.5 M NaCl, residue S486 interacts with the conserved water molecule (C).
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