title: Characterization of the Substrate Specificity of the Dnmt2 Methyltransferase
creator: Meusburger, Madeleine
subject: ddc-570
subject: 570 Life sciences
description: Summary The modification of cytosine to 5-methyl-cytosine within DNA plays an important role in epigenetic processes. In eukaryotes, the methylation reaction is catalyzed by the family of DNA methyltransferases (Dnmts). The Dnmt2 protein is the most conserved and most widely distributed member of the Dnmt family, however, its catalytic function has been an enigma. Structural analysis predicts the enzyme to be a bona fide DNA methyltransferase, though it exhibits only marginal DNA methylating activity in vitro. Recently, however, Dnmt2 was discovered to be a tRNA methyltransferase, catalyzing the formation of 5-methyl-cytosine at position 38 of tRNAAsp within the anticodon stem-loop.  This work contributed to the further characterization of the enzyme in the model organism Drosophila melanogaster. A null mutant was characterized and used for further DNA methylation analysis in comparison to Dnmt2-containing wildtype flies. Dnmt2-dependent DNA methylation was discovered at one defined locus in the fly genome: within the 3´-region of the mini-white marker gene of a mobile element which had inserted into the retrotransposon invader 4.  Further experiments could show that unmodified, in vitro transcribed tRNAs are targets of the Dnmt2 enzyme derived from fly and human, and that other modifications are not a prerequisite for Dnmt2 target recognition. Besides tRNAAsp, tRNAVal was identified as a new target of Dnmt2 in vitro and in vivo. The optimization and versatile application of the DNAzyme technique allowed the quantification of the methylation signal. Furthermore, non-C38 containing tRNAs from various organisms were also found to be methylated by Dnmt2 in vitro. The new methylation site could be mapped to C40 in tRNAPhe from Saccharomyces cerevisiae, and to C40 and C42 in tRNALys from Drosophila melanogaster. The analysis of several catalytic Dnmt2 mutants of human Dnmt2 in combination with various tRNA substrates revealed two different catalytic mechanisms.      
date: 2008
type: Dissertation
type: info:eu-repo/semantics/doctoralThesis
type: NonPeerReviewed
format: application/pdf
identifier: https://archiv.ub.uni-heidelberg.de/volltextserverhttps://archiv.ub.uni-heidelberg.de/volltextserver/8644/1/MadeleineMeusburger_PhD_Thesis.pdf
identifier: DOI:10.11588/heidok.00008644
identifier: urn:nbn:de:bsz:16-opus-86443
identifier:   Meusburger, Madeleine  (2008) Characterization of the Substrate Specificity of the Dnmt2 Methyltransferase.  [Dissertation]     
relation: https://archiv.ub.uni-heidelberg.de/volltextserver/8644/
rights: info:eu-repo/semantics/openAccess
rights: http://archiv.ub.uni-heidelberg.de/volltextserver/help/license_urhg.html
language: eng