Mariko Ariyoshi

Recognition of hemi-methylated DNA by the SRA protein UHRF1 by a base-flipping mechanism

DNA methylation of CpG dinucleotides is an important epigenetic modification of mammalian genomes and is essential for the regulation of chromatin structure, of gene expression and of genome stability. Differences in DNA methylation patterns underlie a wide range of biological processes, such as genomic imprinting, inactivation of the X chromosome, embryogenesis, and carcinogenesis. Inheritance of the epigenetic methylation pattern is mediated by the enzyme DNA methyltransferase 1 (Dnmt1), which methylates newly synthesized CpG sequences during DNA replication, depending on the methylation status of the template strands. The protein UHRF1 (also known as Np95 and ICBP90) recognizes hemi-methylation sites via a SET and RING-associated (SRA) domain and directs Dnmt1 to these sites. Here we report the crystal structures of the SRA domain in free and hemi-methylated DNA-bound states. The SRA domain folds into a globular structure with a basic concave surface formed by highly conserved residues. Binding of DNA to the concave surface causes a loop and an amino-terminal tail of the SRA domain to fold into DNA interfaces at the major and minor grooves of the methylation site. In contrast to fully methylated CpG sites recognized by the methyl-CpG-binding domain, the methylcytosine base at the hemi-methylated site is flipped out of the DNA helix in the SRA–DNA complex and fits tightly into a protein pocket on the concave surface. The complex structure suggests that the successive flip out of the pre-existing methylated cytosine and the target cytosine to be methylated is associated with the coordinated transfer of the hemi-methylated CpG site from UHRF1 to Dnmt1.

Atomic Structure of the RuvC Resolvase: A Holliday Junction-Specific Endonuclease from E. coli

The crystal structure of the RuvC protein, a Holliday junction resolvase from E. coli, has been determined at 2.5 A resolution. The enzyme forms a dimer of 19 kDa subunits related by a dyad axis. Together with results from extensive mutational analyses, the refined structure reveals that the catalytic center, comprising four acidic residues, lies at the bottom of a cleft that nicely fits a DNA duplex. The structural features of the dimer, with a 30 A spacing between the two catalytic centers, provide a substantially defined image of the Holliday junction architecture. The folding topology in the vicinity of the catalytic site exhibits a striking similarity to that of RNAase H1 from E. coli.

Structural basis for recognition of H3K4 methylation status by the DNA methyltransferase 3A ATRX–DNMT3–DNMT3L domain

DNMT3 proteins are de novo DNA methyltransferases that are responsible for the establishment of DNA methylation patterns in mammalian genomes. Here, we have determined the crystal structures of the ATRX–DNMT3–DNMT3L (ADD) domain of DNMT3A in an unliganded form and in a complex with the amino‐terminal tail of histone H3. Combined with the results of biochemical analysis, the complex structure indicates that DNMT3A recognizes the unmethylated state of lysine 4 in histone H3. This finding indicates that the recruitment of DNMT3A onto chromatin, and thereby de novo DNA methylation, is mediated by recognition of the histone modification state by its ADD domain. Furthermore, our biochemical and nuclear magnetic resonance data show mutually exclusive binding of the ADD domain of DNMT3A and the chromodomain of heterochromatin protein 1α to the H3 tail. These results indicate that de novo DNA methylation by DNMT3A requires the alteration of chromatin structure.

Recognition of modification status on a histone H3 tail by linked histone reader modules of the epigenetic regulator UHRF1

Multiple covalent modifications on a histone tail are often recognized by linked histone reader modules. UHRF1 [ubiquitin-like, containing plant homeodomain (PHD) and really interesting new gene (RING) finger domains 1], an essential factor for maintenance of DNA methylation, contains linked two-histone reader modules, a tandem Tudor domain and a PHD finger, tethered by a 17-aa linker, and has been implicated to link histone modifications and DNA methylation. Here, we present the crystal structure of the linked histone reader modules of UHRF1 in complex with the amino-terminal tail of histone H3. Our structural and biochemical data provide the basis for combinatorial readout of unmodified Arg-2 (H3-R2) and methylated Lys-9 (H3-K9) by the tandem tudor domain and the PHD finger. The structure reveals that the intermodule linker plays an essential role in the formation of a histone H3–binding hole between the reader modules by making extended contacts with the tandem tudor domain. The histone H3 tail fits into the hole by adopting a compact fold harboring a central helix, which allows both of the reader modules to simultaneously recognize the modification states at H3-R2 and H3-K9. Our data also suggest that phosphorylation of a linker residue can modulate the relative position of the reader modules, thereby altering the histone H3–binding mode. This finding implies that the linker region plays a role as a functional switch of UHRF1 involved in multiple regulatory pathways such as maintenance of DNA methylation and transcriptional repression.

Crystal Structure of the RuvA-RuvB Complex: A Structural Basis for the Holliday Junction Migrating Motor Machinery

We present the X-ray structure of the RuvA-RuvB complex, which plays a crucial role in ATP-dependent branch migration. Two RuvA tetramers form the symmetric and closed octameric shell, where four RuvA domain IIIs spring out in the two opposite directions to be individually caught by a single RuvB. The binding of domain III deforms the protruding beta hairpin in the N-terminal domain of RuvB and thereby appears to induce a functional and less symmetric RuvB hexameric ring. The model of the RuvA-RuvB junction DNA ternary complex, constructed by fitting the X-ray structure into the averaged electron microscopic images of the RuvA-RuvB junction, appears to be more compatible with the branch migration mode of a fixed RuvA-RuvB interaction than with a rotational interaction mode.

Structure of the small ubiquitin-like modifier (SUMO)-interacting motif of MBD1-containing chromatin-associated factor 1 bound to SUMO-3

Post-translational modification by small ubiquitin-like modifier (SUMO) proteins has been implicated in the regulation of a variety of cellular events. The functions of sumoylation are often mediated by downstream effector proteins harboring SUMO-interacting motifs (SIMs) that are composed of a hydrophobic core and a stretch of acidic residues. MBD1-containing chromatin-associated factor 1 (MCAF1), a transcription repressor, interacts with SUMO-2/3 and SUMO-1, with a preference for SUMO-2/3. We used NMR spectroscopy to solve the solution structure of the SIM of MCAF1 bound to SUMO-3. The hydrophobic core of the SIM forms a parallel β-sheet pairing with strand β2 of SUMO-3, whereas its C-terminal acidic stretch seems to mediate electrostatic interactions with a surface area formed by basic residues of SUMO-3. The significance of these electrostatic interactions was shown by mutations of both SUMO-3 and MCAF1. The present structural and biochemical data suggest that the acidic stretch of the SIM of MCAF1 plays an important role in the binding to SUMO-3.

Functional analyses of the domain structure in the Holliday junction binding protein RuvA.

BACKGROUND Homologous recombination is crucial for genetic diversity and repairing damaged chromosomes. In Escherichia coli cells, the RuvA, RuvB and RuvC proteins participate in the processing of an important intermediate, the Holliday junction. The RuvA-RuvB protein complex facilitates branch migration of the junction, depending on ATP hydrolysis. The atomic structure of RuvA should enable critical questions to be addressed about its specific interactions with the Holliday junction and the RuvB protein. RESULTS The crystal structure of RuvA shows the tetrameric molecules with a fourfold axis at the center. Each subunit consists of three distinct domains, some of which contain important secondary structure elements for DNA binding. Together with the detailed structural information, the biochemical assays of various mutant RuvA proteins and domains, isolated by partial proteolysis, allowed us to define the functional roles of these domains in Holliday junction binding and the RuvB interaction. CONCLUSIONS The RuvA molecule is formed by four identical subunits, each with three domains, I, II and III. The locations of the putative DNA-binding motifs define an interface between the DNA and the Holliday junction. Domain III is weakly attached to the core region, comprising domains I and II; the core domains can form a tetramer in the absence of domain III. Functional analyses of the mutant proteins and the partial digestion products, including Holliday junction binding and branch-migration assays, revealed that domain III and the preceding loop are crucial for RuvB binding and branch migration, although this region is not required for the junction-DNA binding.

Structural Basis of the Versatile DNA Recognition Ability of the Methyl-CpG Binding Domain of Methyl-CpG Binding Domain Protein 4

Background: Methyl-CpG binding domain 4 (MBD4) is a DNA glycosylase that excises mismatched bases generated in methylated CpG sequences. Results: We report the biochemical and structural properties of the methyl-CpG binding domain of MBD4 (MBDMBD4). Conclusion: MBDMBD4 recognizes a wide range of 5-methylcytosine modifications via an extensive hydration network. Significance: This study provides new insight into the structural mechanism of the broad base recognition that is unique to MBDMBD4. The methyl-CpG binding domain (MBD) protein MBD4 participates in DNA repair as a glycosylase that excises mismatched thymine bases in CpG sites and also functions in transcriptional repression. Unlike other MBD proteins, MBD4 recognizes not only methylated CpG dinucleotides (5mCG/5mCG) but also T/G mismatched sites generated by spontaneous deamination of 5-methylcytosine (5mCG/TG). The glycosylase activity of MBD4 is also implicated in active DNA demethylation initiated by the deaminase-catalyzed conversion of 5-methylcytosine to thymine. Here, we report the crystal structures of the MBD of MBD4 (MBDMBD4) complexed with 5mCG/5mCG and 5mCG/TG. The crystal structures show that the DNA interface of MBD4 has flexible structural features and harbors an extensive water network that supports its dual base specificities. Combined with the results of biochemical analyses, the crystal structure of MBD4 bound to 5-hydroxymethylcytosine further demonstrates that MBDMBD4 is able to recognize a wide range of 5-methylcytosine modifications through the unique water network. The versatile base recognition ability of MBDMBD4 implies multifunctional roles for MBD4 in the regulation of dynamic DNA methylation patterns coupled with deamination and/or oxidation of 5-methylcytosine.

Molecular Basis for SUMOylation-dependent Regulation of DNA Binding Activity of Heat Shock Factor 2

Heat shock factor 2 (HSF2) is a member of a vertebrate transcription factor family for genes of heat shock proteins and is involved in the regulation of development and cellular differentiation. The DNA binding property of HSF2 is modulated by the post-translational modification of a specific lysine residue in its DNA binding domain by small ubiquitin-like modifier (SUMO), but the consequences of SUMOylation and its underlying molecular mechanism remain unclear. Here we show the inhibitory effect of SUMOylation on the interaction between HSF2 and DNA based on biochemical analysis using isolated recombinant HSF2. NMR study of the SUMOylated DNA binding domain of HSF2 indicates that the SUMO moiety is flexible with respect to the DNA binding domain and has neither a noncovalent interface with nor a structural effect on the domain. Combined with data from double electron-electron resonance and paramagnetic NMR relaxation enhancement experiments, these results suggest that SUMO attachment negatively modulates the formation of the protein-DNA complex through a randomly distributed steric interference.

Structural basis for regulation of poly-SUMO chain by a SUMO-like domain of Nip45

Post‐translational modification by small ubiquitin‐like modifier (SUMO) provides an important regulatory mechanism in diverse cellular processes. Modification of SUMO has been shown to target proteins involved in systems ranging from DNA repair pathways to the ubiquitin‐proteasome degradation system by the action of SUMO‐targeted ubiquitin ligases (STUbLs). STUbLs recognize target proteins modified with a poly‐SUMO chain through their SUMO‐interacting motifs (SIMs). STUbLs are also associated with RENi family proteins, which commonly have two SUMO‐like domains (SLD1 and SLD2) at their C terminus. We have determined the crystal structures of SLD2 of mouse RENi protein, Nip45, in a free form and in complex with a mouse E2 sumoylation enzyme, Ubc9. While Nip45 SLD2 shares a β‐grasp fold with SUMO, the SIM interaction surface conserved in SUMO paralogues does not exist in SLD2. Biochemical data indicates that neither tandem SLDs or SLD2 of Nip45 bind to either tandem SIMs from either mouse STUbL, RNF4 or to those from SUMO‐binding proteins, whose interactions with SUMO have been well characterized. On the other hand, Nip45 SLD2 binds to Ubc9 in an almost identical manner to that of SUMO and thereby inhibits elongation of poly‐SUMO chains. This finding highlights a possible role of the RENi proteins in the modulation of Ubc9‐mediated poly‐SUMO formation. Proteins 2010.