Structural insight into maintenance methylation by mouse DNA methyltransferase 1 (Dnmt1)

Abstract

Methylation of cytosine in DNA plays a crucial role in development through inheritable gene silencing. The DNA methyltransferase Dnmt1 is responsible for the propagation of methylation patterns to the next generation via its preferential methylation of hemimethylated CpG sites in the genome; however, how Dnmt1 maintains methylation patterns is not fully understood. Here we report the crystal structure of the large fragment (291-1620) of mouse Dnmt1 and its complexes with cofactor S-adenosyl-L-methionine and its product S-adenosyl-L-homocystein. Notably, in the absence of DNA, the N-terminal domain responsible for targeting Dnmt1 to replication foci is inserted into the DNA-binding pocket, indicating that this domain must be removed for methylation to occur. Upon binding of S-adenosyl-L-methionine, the catalytic cysteine residue undergoes a conformation transition to a catalytically competent position. For the recognition of hemimethylated DNA, Dnmt1 is expected to utilize a target recognition domain that overhangs the putative DNA-binding pocket. Taking into considerations the recent report of a shorter fragment structure of Dnmt1 that the CXXC motif positions itself in the catalytic pocket and prevents aberrant de novo methylation, we propose that maintenance methylation is a multistep process accompanied by structural changes.

Fig. 1.

Multidomain structure of mouse Dnmt1(291–1620). (A) Replication foci targeting sequence (RFTS), zincfinger-like motif (CXXC), bromo-associated homology domain 1 (BAH1) and 2 (BAH2), and catalytic domain are schematically illustrated. The catalytic domain comprising 10 conserved motifs (I ∼ X), and target recognition domain (TRD) between the motifs VIII and IX. The numbers of the amino acid residues are indicated. (B) Ribbon model of mouse Dnmt1(291–1620). Around the C-terminal catalytic domain (blue), the other domains including the RFTS (magenta), CXXC motif (cyan), and two BAH domains BAH1 (green) and BAH2 (orange) are shown. Four zinc ions are shown in red spheres. All of the zinc ions are in a motif similar to Zn-finger motif (Fig. S10). The KG-repeat (1112–1124) linker connecting the N-terminal region and the C-terminal catalytic domain is in a flexible structure as the density map showed disorder.

Fig. 2.

The RFTS domain plugs into the DNA-binding pocket. (A) The RFTS domain is positioned in the DNA-binding pocket of Dnmt1 and stabilized by several hydrogen bonds in the catalytic domain. (B) The linker (brown) between the RFTS domain (deep red) and the CXXC motif (light blue) interacts with the catalytic domain (dark blue) by hydrophobic forces to maintain the position of the PCQ loop (see Fig. S7). C. Fitting to Arrhenius equation of the DNA methylation activity of Dnmt1(291–1620) (cyan diamonds) containing the RFTS domain and Dnmt1(602–1620) (magenta squares) lacking the RFTS domain. The activation energies of Dnmt1(291–1620) and Dnmt1(602–1620) were calculated to be 110 and 30 kJ/mol, respectively.

Fig. 3.

Model of conformational changes that are predicted to be induced by DNA binding. Superimposition of the free-form structure including the RFTS domain (blue, PBD ID code 3av4) and the complex with unmethylated DNA structure (yellow, PDB ID code 3pt6). The CXXC motifs (dark blue) and the linker connecting the RFTS domain and CXXC motif (light blue) are indicated in deep colors, and the rest sequences are in pale colors. The RFTS domain (pale gray) and unmethylated DNA (pale yellow) are shown in surface model. The sequences involved in the interaction of the linker connecting the RFTS domain and CXXC motif with the PCQ loop at the catalytic center are magnified.

Fig. 4.

AdoMet turns the side chain of the cysteine in the PCQ loop toward the target cytosine. (A) Superimposition of the PCQ loop of Dnmt1 in its free form (green), with AdoH (orange), and with AdoMet (blue). (B) Binding of AdoMet to the catalytic site induces the side chain of C1229 to turn toward the target cytosine (Left). C1229 is expected to bind to C6 and the methyl-group of AdoMet binds to C5, of the cytosine (red arrows). After transfer of the methyl-group to the fifth position of cytosine, C1229 turns back to the inactive AdoH-binding form (Right). The position of cytosine was taken from PDB ID code 5mht, the structure of M.HhaI complex with methylated DNA and AdoH.

Fig. 5.

The TRD of Dnmt1 overhangs the putative DNA-binding pocket. (A) Superimposition of the catalytic domain of Dnmt1 with M.HhaI. The positions of the TRD of Dnmt1 (light blue) and the hemimethylated double-stranded DNA fitted form of the 10 motifs (blue) of the catalytic domain of Dnmt1(291–1620) superimposed with M.HhaI (PDB ID code 5mht) (pink) are shown. (B) DNA-fitted model of Dnmt1(291–1620) showing that W1512 is close to the methylated cytosine base of the hemimethylated DNA when the methylated cytosine (5MC) stays inside the double-stranded DNA, whereas W1500 is close to when the 5MC is flipped out.