Biochemical characterization and molecular evolution of the Dnmt2 RNA/DNA metyltransferase

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Dnmt2 enzymes are strongly conserved and found in species ranging from Schizosaccharomyces pombe to human, suggesting a very important role for cellular homeostasis. However, most of dnmt2 knock-out organism originally lacked an apparent phenotype such that Dnmt2's biological function remained unknown. Despite their sequence and structural similarity to DNA methyltransferases, it was discovered in 2006 that Dnmt2 is a highly active RNA methyltransferase capable of methylating the C38 position of the tRNA-Asp. Based on extensive preliminary work and in close cooperation with the other groups of FOR1082, we plan to investigate here three important features of the mechanism, function and evolution of Dnmt2. Subject area 1 has the aim to improve our understanding of the structure and mechanism of action of Dnmt2 in particular with respect to RNA and possibly DNA substrate recognition. We will study questions like: How does Dnmt2 recognize its RNA targets? Are there Dnmt2 proteins with strong in vitro DNA methylation activity or can such variants be generated? Does Dnmt2 methylate DNA with unusual structures? Subject area 2 aims to investigate the biological function of the tRNA-Asp C38 methylation catalyzed by Dnmt2. This includes experiments which address questions like: Does C38 methylation contribute to the recognition of tRNA-Asp by Aspartyl-tRNA-synthetase? Does C38 methylation influence the rate of mischarging this tRNA? Does C38 methylation influence the general translation process in vivo? Are there changes in the proteome of Dnmt2 knock-out cells that can be correlated with the effect of the C38 methylation on tRNA charging and/or general translation? Subject area 3 studies the molecular evolution of Dnmt2 as an example of the evolution of a multifunctional enzyme. Main questions are: What is the evolutionary history of Dnmt2? Can we reconcile and experimentally follow its change in substrate specificity from DNA to tRNA? What is the role of the only Dnmt2 enzyme found in a bacterial species? Can we experimentally influence the specificity of Dnmt2 and eventually predict function from sequence?