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 RNAs that act like guide RNAs in vitro. The 5'-half of tRNAAspwas able to efficiently guide methylation toward a single stranded tRNA fragment as would result from tRNA cleavage by tRNA specific nucleases. In a more artificial setting, a composite system of guide RNAs could ultimately be engineered to enable the enzyme to perform cytidine methylation on single stranded DNA in vitro.
a Institute of Pharmacy and Biochemistry, Johannes Gutenberg University Mainz , Mainz , Germany.
Steffen
S
Kaiser
b Institute of Biochemistry, Faculty of Chemistry, University Stuttgart , Stuttgart , Germany.
Tomasz P
TP
Jurkowski
a Institute of Pharmacy and Biochemistry, Johannes Gutenberg University Mainz , Mainz , Germany.
Stefanie
S
Kellner
a Institute of Pharmacy and Biochemistry, Johannes Gutenberg University Mainz , Mainz , Germany.
Dirk
D
Schneider
b Institute of Biochemistry, Faculty of Chemistry, University Stuttgart , Stuttgart , Germany.
Albert
A
Jeltsch
a Institute of Pharmacy and Biochemistry, Johannes Gutenberg University Mainz , Mainz , Germany.
Mark
0000-0002-0154-0928
M
Helm
D000818:Animals; D003596:Cytosine / Q000378:metabolism; D004247:DNA / Q000378:metabolism*; D004248:DNA (Cytosine-5-)-Methyltransferases / Q000378:metabolism*; D019175:DNA Methylation; D006801:Humans; D008745:Methylation; D051379:Mice; D009690:Nucleic Acid Conformation; D012343:RNA, Transfer / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D013329:Structure-Activity Relationship
D003596:Cytosine; D004247:DNA; D012343:RNA, Transfer; D004248:DNA (Cytosine-5-)-Methyltransferases; C112218:TRDMT1 protein, human
5-methylcytosine; DNA; Dnmt2; RNA methylation; RNA modification; enzyme kinetics; modification pathway crosstalk; tRNA