RNA-Substrate Sepcificity of Dnmt2

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Dnmt2 is a well aligned member of the eukaryotic family of DNA methyltransferases, but it efficiently catalyzes tRNA methylation. The role of the resulting ribo‐5‐methylcytidine (ribo‐m5C) at position 38 of different tRNAs is poorly understood at present, while the desoxy‐m5C residues formed by Dnmt1 and Dnmt3 are thought to be classical epigenetic marks. While the catalytic mechanism of nucleic acid modification between the two types of nucleic acid MTases is similar, their functions seem to strongly diverge, yet DNA methylation may still be part of the Dnmt2 activity. In this backdrop, the project addresses the nucleic acid substrate recognition by Dnmt2 as a characteristic biochemical feature that is informative in the comparison of Dnmt2 with other MTases. Our analysis so far shows that most of the tRNA structure is required in addition to a consensus motif comprising seven nucleotides in the anticodon domain. A refined analysis will be performed by a combinatorial approach based on catalytic alkylation of Dnmt2 substrates with a cofactor analogue. The transferred alkylgroup contains a terminal alkyne, which can be conjugated to biotin by click chemistry for physical separation of Dnmt2 substrates. This is followed by deep sequencing or by amplification for a renewed selection. In another line of investigation, we have found that tRNAs containing stretches of up to ten deoxynucleotides at and around the methylation site are efficient substrates of Dnmt2. We will investigate methylation activity of hybrid substrates with maximized DNA content, to assess the degree of biochemical similarity of Dnmt2 to other Dnmt enzymes and further MTases. Further nucleic acid hybrids with varying contents of RNA and DNA, in which both types of nucleic acids are either covalently linked, or assembled by annealing, will be developed into an in vitro system for guided methylation by Dnmt2. Accompanying analytics of cytidine methylation by LC‐MS and selective chemical modification will be developed.