Mechanism and biological role of Dnmt2 in Nucleic Acid Methylation
ABSTRACT A group of homologous nucleic acid modification enzymes called Dnmt2, Trdmt1, Pmt1, DnmA, and Ehmet in different model organisms catalyze the transfer of a methyl group from the cofactor S-adenosyl-methionine (SAM) to the carbon-5 of cytosine residues. Originally considered as DNA MTases, these enzymes were shown to be tRNA methyltransferases about a decade ago. Between the presumed involvement in DNA modification-related epigenetics, and the recent foray into the RNA modification field, significant progress has characterized Dnmt2-related research. Here, we review this progress in its diverse facets including molecular evolution, structural biology, biochemistry, chemical biology,…
The RNA methyltransferase Dnmt2 methylates DNA in the structural context of a tRNA
The amino acid sequence of Dnmt2 is very similar to the catalytic domains of bacterial and eukaryotic DNA-(cytosine 5)-methyltransferases, but it efficiently catalyzes tRNA methylation, while its DNA methyltransferase activity is the subject of controversial reports with rates varying between zero and very weak. By using composite nucleic acid molecules as substrates, we surprisingly found that DNA fragments, when presented as covalent DNA-RNA hybrids in the structural context of a tRNA, can be more efficiently methylated than the corresponding natural tRNA substrate. Furthermore, by stepwise development of tRNAAsp, we showed that this natural Dnmt2 substrate could be engineered to employ R…
The Dnmt2 RNA methyltransferase homolog of Geobacter sulfurreducens specifically methylates tRNA-Glu
Dnmt2 enzymes are conserved in eukaryotes, where they methylate C38 of tRNA-Asp with high activity. Here, the activity of one of the very few prokaryotic Dnmt2 homologs from Geobacter species (GsDnmt2) was investigated. GsDnmt2 was observed to methylate tRNA-Asp from flies and mice. Unexpectedly, it had only a weak activity toward its matching Geobacter tRNA-Asp, but methylated Geobacter tRNA-Glu with good activity. In agreement with this result, we show that tRNA-Glu is methylated in Geobacter while the methylation is absent in tRNA-Asp. The activities of Dnmt2 enzymes from Homo sapiens, Drosophila melanogaster, Schizosaccharomyces pombe and Dictyostelium discoideum for methylation of the …
Cytosine methylation of tRNA-Asp by DNMT2 has a role in translation of proteins containing poly-Asp sequences
AbstractThe Dnmt2 RNA methyltransferase catalyses the methylation of C38 in the anticodon loop of tRNA-Asp, but the molecular role of this methylation is unknown. Here, we report that mouse aspartyl-tRNA synthetase shows a four to fivefold preference for C38-methylated tRNA-Asp. Consistently, a 30% reduced charging level of tRNA-Asp was observed in Dnmt2 knockout (KO) murine embryonic fibroblast cells. Gene expression analysis with fluorescent reporter proteins fused to an N-terminal poly-Asp sequence showed that protein synthesis of poly-Asp-tagged reporter proteins was reduced in Dnmt2 KO cells as well. The same effect was observed with endogenous proteins containing poly-Asp sequences, i…
Mapping the tRNA binding site on the surface of human DNMT2 methyltransferase.
The DNMT2 enzyme methylates tRNA-Asp at position C38. Because there is no tRNA–Dnmt2 cocrystal structure available, we have mapped the tRNA binding site of DNMT2 by systematically mutating surface-exposed lysine and arginine residues to alanine and studying the tRNA methylation activity and binding of the corresponding variants. After mutating 20 lysine and arginine residues, we identified eight of them that caused large (>4-fold) decreases in catalytic activity. These residues cluster within and next to a surface cleft in the protein, which is large enough to accommodate the tRNA anticodon loop and stem. This cleft is located next to the binding pocket for the cofactor S-adenosyl-l-methion…