Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
Comparative Study
. 2001 Jan 15;29(2):439-48.
doi: 10.1093/nar/29.2.439.

Structure of human DNMT2, an enigmatic DNA methyltransferase homolog that displays denaturant-resistant binding to DNA

A Dong  1 , J A Yoder, X Zhang, L Zhou, T H Bestor, X Cheng
Affiliations
Comparative Study

Structure of human DNMT2, an enigmatic DNA methyltransferase homolog that displays denaturant-resistant binding to DNA

A Dong et al. Nucleic Acids Res. .

Abstract

DNMT2 is a human protein that displays strong sequence similarities to DNA (cytosine-5)-methyltransferases (m(5)C MTases) of both prokaryotes and eukaryotes. DNMT2 contains all 10 sequence motifs that are conserved among m(5)C MTases, including the consensus S:-adenosyl-L-methionine-binding motifs and the active site ProCys dipeptide. DNMT2 has close homologs in plants, insects and Schizosaccharomyces pombe, but no related sequence can be found in the genomes of Saccharomyces cerevisiae or Caenorhabditis elegans. The crystal structure of a deletion mutant of DNMT2 complexed with S-adenosyl-L-homocysteine (AdoHcy) has been determined at 1.8 A resolution. The structure of the large domain that contains the sequence motifs involved in catalysis is remarkably similar to that of M.HHAI, a confirmed bacterial m(5)C MTase, and the smaller target recognition domains of DNMT2 and M.HHAI are also closely related in overall structure. The small domain of DNMT2 contains three short helices that are not present in M.HHAI. DNMT2 binds AdoHcy in the same conformation as confirmed m(5)C MTases and, while DNMT2 shares all sequence and structural features with m(5)C MTases, it has failed to demonstrate detectable transmethylase activity. We show here that homologs of DNMT2, which are present in some organisms that are not known to methylate their genomes, contain a specific target-recognizing sequence motif including an invariant CysPheThr tripeptide. DNMT2 binds DNA to form a denaturant-resistant complex in vitro. While the biological function of DNMT2 is not yet known, the strong binding to DNA suggests that DNMT2 may mark specific sequences in the genome by binding to DNA through the specific target-recognizing motif.

PubMed Disclaimer

Figures

Figure 1
Figure 1
Alignment of Dnmt2 homologs. The slashed lines indicate sequences missing from the available ESTs and dashed lines indicate gaps introduced to optimize alignments. The human DNMT2 residue numbering is shown above the sequence alignment. Amino acids highlighted are either invariant (white against black) among the proteins or similar (shaded), as defined by the following groupings: V, L, I and M; F, Y and W; K and R; E and D; Q and N; S and T; A, G and P. The secondary structural elements of DNMT2 (helices A–X and strands 1–11) and conserved sequence motifs are labeled. Complete Dnmt2 sequences are available in GenBank for human (AAC39764), mouse (AAC53529) and S.pombe (CAA57824). The two Dnmt2 sequences of D.melanogaster currently deposited in GenBank, AAF03835 and AAF53163, contain a single exon and lack the consensus motif I. We used the sequence reported by Tweedie et al. (24), which includes an additional upstream exon and contains motif I. The A.thaliana sequence was assembled from genomic sequence AC006601 between nucleotides 29989 and 27859. Two of the eight splicing junctions are confirmed by EST sequences. The X.laevis Dnmt2 sequence was assembled from two overlapping ESTs (AW639976 and AW636415) and lacks the C-terminus of the protein. The D.rerio sequence was assembled by jointing four ESTs (AI331437, AI618465, AI331180 and AI331323). The B.mori sequence was assembled from three ESTs (AU005443, AV399933 and AV400288). Shown in dashed overline and labeled V8 are the sequences (from 191 to 237) deleted to make a version of DNMT2 that could be crystallized. Note that these sequences are normally absent from the D.melanogaster, S.pombe and B.mori Dnmt2 homologs.
Figure 2
Figure 2
Ribbon (42) diagrams of the DNMT2Δ47–AdoHcy complex. (A) Front view. (B) Right side view (the point of view of an observer from the right side of Fig. 2A). Helices and loops are colored red (unique helices of DNMT2 gray), strands in green, the PC loop in light blue and the TRD loop in green. The bound AdoHcy and CFT of the TRD are shown as balls, with carbon atoms in black, nitrogen atoms in light blue, oxygen atoms in magenta and sulfur atoms in orange. (C) Superimposition of DNMT2Δ47, colored yellow (residues 1–188) and blue (residues 248–391), and M.HhaI (pdb 1HMY), colored green. (D) Topology of DNMT2.
Figure 3
Figure 3
Detailed plots of interactions centered on (A) Tyr10 of motif I and (B) Asn158 of motif VIII. (C) Superimposition of the TRD loop containing Cys292-Phe293-Thr294 of DNMT2 (blue) and Thr250-Leu251 of M.HhaI (green).
Figure 4
Figure 4
(A) AdoHcy is shown in stick form and is superimposed on a Fo – Fc omit electron density map. (B) Solvent-accessible molecular GRASP surface (30) of the electrostatic potential of DNMT2Δ47. The surface is colored blue for positive (10 kT), red for negative (–10 kT) and white for neutral, where k is the Boltzmann constant and T is the temperature. (C) Model of DNMT2Δ47–AdoHcy bound to DNA. The DNA model is from the structure of the M.HhaI–AdoHcy–DNA complex (pdb 3MHT; 31).
Figure 5
Figure 5
(A) Formation of denaturant-resistant DNMT2–DNA complexes. DNMT2 (4 µg/ml) was incubated at 37°C with oligonucleotides (1 µg/ml) in 20 mM Tris–HCl pH 8.0, 1 mM EDTA, 25 mM NaCl, 0.2 mM PMSF, 0.5 mM DTT and 40 µM AdoMet. Reactions were terminated at the indicated times by addition of SDS to 2% and glycerol to 12% and heating to 65°C for 10 min, subjected to 6% SDS–PAGE, transferred to nitrocellulose and autoradiographed. Oligonucleotides were of sequence 5′-CCTTTACAAATTTCCAATGCNNNNFGNNNNNNNNFNNNNNNNNNFGNNNNCCTGAAAAAAGACTAATTAAATTCATGGTA-3′, where N is a random base. The synthesis, radioactive labeling and purification of FdC oligonucleotides have been described (16). The control oligonucleotide duplexes were not methylated, while FdC duplexes were hemimethylated (16). Methylation status did not affect formation of complexes (data not shown). Lanes headed F contain FdC oligonucleotides and lanes headed C contain control oligonucleotides with C replacing FdC. (B) Effect of ES cell lysates on formation of DNMT2–DNA complexes. Aliquots of 70 µg ES cell lysate were incubated with 0.1 µg internally labeled FdC or control oligonucleotide in 100 µl of 20 mM Tris–HCl pH 7.4, 10% glycerol, 1 mM EDTA, 0.5 mM DTT, 0.2 mM PMSF and 40 µM AdoMet. Where indicated, purified DNMT2 (0.4 µg) was added at the beginning of the reaction. Reactions were terminated after 3 h at 37°C by addition of SDS to 2% and heating to 65°C for 10 min, subjected to 6% SDS–PAGE, transferred to nitrocellulose and autoradiographed. (C) Inhibition of DNMT2–DNA complex formation by nucleoside triphosphates. Incubation of DNMT2 with FdC oligonucleotide and 1 mM ATP, GTP or ATPγS prevented complex formation but had no effect on the reaction of M.SssI with the FdC oligonucleotide. The inhibitory effect could be largely overcome by addition of equimolar amounts of AdoMet (lanes 5 and 6). In all cases electrophoresis was halted just after unbound oligonucleotides migrated off the lower end of the gel.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Bestor T.H. (2000) The DNA methyltransferases of mammals. Hum. Mol. Genet., 9, 2395–2402. - PubMed
    1. Lei H., Oh,S.P., Okano,M., Juttermann,R., Goss,K.A., Jaenisch,R. and Li,E. (1996) De novo DNA cytosine methyltransferase activities in mouse embryonic stem cells. Development, 122, 3195–3205. - PubMed
    1. Okano M., Bell,D.W., Haber,D.A. and Li,E. (1999) DNA methyltransferases Dnmt3a and Dnmt3b are essential for de novo methylation and mammalian development. Cell, 99, 247–257. - PubMed
    1. Xu G.-L., Bestor,T.H., Bourc’his,D., Hsieh,C.-L., Tommerup,N., Bugge,M., Hulten,M., Qu,X., Russo,J.J. and Viegas-Pequignot,E. (1999) Chromosome instability and immunodeficiency syndrome caused by mutations in a DNA methyltransferase gene. Nature, 402, 187–191. - PubMed
    1. Okano M., Xie,S. and Li,E. (1998) Dnmt2 is not required for de novo and maintenance methylation of viral DNA in embryonic stem cells. Nucleic Acids Res., 26, 2536–2540. - PMC - PubMed

Publication types

MeSH terms

Substances

Associated data