A dual flip-out mechanism for 5mC recognition by the Arabidopsis SUVH5 SRA domain and its impact on DNA methylation and H3K9 dimethylation in vivo

Structural Biology Program, Memorial Sloan-Kettering Cancer Center, New York, New York 10065, USA.
Genes & development (Impact Factor: 10.8). 01/2011; 25(2):137-52. DOI: 10.1101/gad.1980311
Source: PubMed


Cytosine DNA methylation is evolutionarily ancient, and in eukaryotes this epigenetic modification is associated with gene silencing. Proteins with SRA (SET- or RING-associated) methyl-binding domains are required for the establishment and/or maintenance of DNA methylation in both plants and mammals. The 5-methyl-cytosine (5mC)-binding specificity of several SRA domains have been characterized, and each one has a preference for DNA methylation in different sequence contexts. Here we demonstrate through mobility shift assays and calorimetric measurements that the SU(VAR)3-9 HOMOLOG 5 (SUVH5) SRA domain differs from other SRA domains in that it can bind methylated DNA in all contexts to similar extents. Crystal structures of the SUVH5 SRA domain bound to 5mC-containing DNA in either the fully or hemimethylated CG context or the methylated CHH context revealed a dual flip-out mechanism where both the 5mC and a base (5mC, C, or G, respectively) from the partner strand are simultaneously extruded from the DNA duplex and positioned within binding pockets of individual SRA domains. Our structure-based in vivo studies suggest that a functional SUVH5 SRA domain is required for both DNA methylation and accumulation of the H3K9 dimethyl modification in vivo, suggesting a role for the SRA domain in recruitment of SUVH5 to genomic loci.

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Available from: Julie A Law, Nov 21, 2014
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    • "Most algal KMT1 proteins show high sequence similarity to land plant KMT1 polypeptides, both within the SET domain and in the surrounding regions known as the Pre-SET and Post-SET motifs (Fig. 1). Additionally, all algal sequences contain an SRA (SET and RING associated) domain (Fig. 1), which recognizes the methylation status of CG and CHH DNA sequences (where H = A, T, or C) (Rajakumara et al., 2011). Land plant KMT1 proteins have been reported to fall into several distinct subgroups, indicative of functional diversification (Casas-Mollano et al., 2007; Huang et al., 2011). "
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    ABSTRACT: Microalgae exhibit enormous diversity and can potentially contribute to the production of biofuels and high value compounds. However, for most species, our knowledge of their physiology, metabolism, and gene regulation is fairly limited. In eukaryotes, gene silencing mechanisms play important roles in both the reversible repression of genes that are required only in certain contexts and the suppression of genome invaders such at transposons. The recent sequencing of several algal genomes is providing insights into the complexity of these mechanisms in microalgae. Collectively, glaucophyte, red, and green microalgae contain the machineries involved in repressive histone H3 lysine methylation, DNA cytosine methylation, and RNA interference. However, individual species often only have subsets of these gene silencing mechanisms. Moreover, current evidence suggests that algal silencing systems function in transposon and transgene repression but their role(s) in gene regulation or other cellular processes remains virtually unexplored, hindering rational genetic engineering efforts. Copyright © 2014 Elsevier Ltd. All rights reserved.
    Bioresource Technology 10/2014; 184. DOI:10.1016/j.biortech.2014.10.119 · 4.49 Impact Factor
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    • "VAST and DALI searches also revealed that the C-terminal domain of AbaSI is structurally similar to the SET and RING-finger associated (SRA) domains of Arabidopsis SUVH5 (44), human and mouse UHRF1 (45–47), and the N-terminal DNA-binding domain of MspJI (48) (Supplementary Figure S5). In AbaSI, this domain contains eight β-strands (in the order 10, 11, 12, 15, 14, 13, 9 and 8) that together roughly form one twisted β-sheet resembling an arch (the ‘beta-arch’). "
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    ABSTRACT: AbaSI, a member of the PvuRts1I-family of modification-dependent restriction endonucleases, cleaves deoxyribonucleic acid (DNA) containing 5-hydroxymethylctosine (5hmC) and glucosylated 5hmC (g5hmC), but not DNA containing unmodified cytosine. AbaSI has been used as a tool for mapping the genomic locations of 5hmC, an important epigenetic modification in the DNA of higher organisms. Here we report the crystal structures of AbaSI in the presence and absence of DNA. These structures provide considerable, although incomplete, insight into how this enzyme acts. AbaSI appears to be mainly a homodimer in solution, but interacts with DNA in our structures as a homotetramer. Each AbaSI subunit comprises an N-terminal, Vsr-like, cleavage domain containing a single catalytic site, and a C-terminal, SRA-like, 5hmC-binding domain. Two N-terminal helices mediate most of the homodimer interface. Dimerization brings together the two catalytic sites required for double-strand cleavage, and separates the 5hmC binding-domains by ∼70 Å, consistent with the known activity of AbaSI which cleaves DNA optimally between symmetrically modified cytosines ∼22 bp apart. The eukaryotic SET and RING-associated (SRA) domains bind to DNA containing 5-methylcytosine (5mC) in the hemi-methylated CpG sequence. They make contacts in both the major and minor DNA grooves, and flip the modified cytosine out of the helix into a conserved binding pocket. In contrast, the SRA-like domain of AbaSI, which has no sequence specificity, contacts only the minor DNA groove, and in our current structures the 5hmC remains intra-helical. A conserved, binding pocket is nevertheless present in this domain, suitable for accommodating 5hmC and g5hmC. We consider it likely, therefore, that base-flipping is part of the recognition and cleavage mechanism of AbaSI, but that our structures represent an earlier, pre-flipped stage, prior to actual recognition.
    Nucleic Acids Research 06/2014; 42(12). DOI:10.1093/nar/gku497 · 9.11 Impact Factor
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    • "In this complex, both 5hmC and the unmethylated cytosine located on the opposite strands flip out of the duplex, and both flipped-out bases were bound by UHRF2-SRA (Figure 4B). This binding feature closely resembles that observed in the complex of the SRA domain of Arabidopsis SUVH5 with hemimethylated CG DNA, where both methylated and unmethylated cytosines on the opposite strands flip out and each base was bound to an SRA domain (Rajakumara et al., 2011). Superimposing UHRF1-SRA with the two UHRF2-SRA molecules in the present structure reveals steric clashes between the NKR loops of the two superimposed UHRF1-SRA molecules (Figure 4B). "
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    ABSTRACT: Methylated cytosine of CpG dinucleotides in vertebrates may be oxidized by Tet proteins, a process that can lead to DNA demethylation. The predominant oxidation product, 5-hydroxymethylcytosine (5hmC), has been implicated in embryogenesis, cell differentiation, and human diseases. Recently, the SRA domain of UHRF2 (UHRF2-SRA) has been reported to specifically recognize 5hmC, but how UHRF2 recognizes this modification is unclear. Here we report the structure of UHRF2-SRA in complex with a 5hmC-containing DNA. The structure reveals that the conformation of a phenylalanine allows the formation of an optimal 5hmC binding pocket, and a hydrogen bond between the hydroxyl group of 5hmC and UHRF2-SRA is critical for their preferential binding. Further structural and biochemical analyses unveiled the role of SRA domains as a versatile reader of modified DNA, and the knowledge should facilitate further understanding of the biological function of UHRF2 and the comprehension of DNA hydroxymethylation in general.
    Molecular cell 05/2014; 54(5). DOI:10.1016/j.molcel.2014.04.003 · 14.02 Impact Factor
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