Characterization of plant associated AlkB-like demethylases

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The AlkB protein from E. coli was some years ago shown to be an iron- and 2-oxoglutarate dependent oxygenase which catalyses the reversal of certain methyl lesions, e. g. 1-methyladenine, in DNA and RNA. In the AlkB reaction, the deleterious methyl group is first oxidised, and then spontaneously released as formaldehyde, thus regenerating the lesion-free base. Vertebrates have eight different AlkB homologues ABH), denoted ABH1-8. Two of these, ABH2 and ABH3, have been shown to reverse lesions on nucleic a cids, whereas the function of the remaining six is still elusive. Counterparts of most of the vertebrate ABH proteins are found in plants, and the proposed project will mainly focus on three different ABH proteins from the model plant Arabidopsis thaliana , denoted AtABH2/3, AtABH5/7, and AtABH8, where we have preliminary data suggesting that they are involved in DNA repair, epigenetic regulation, and tRNA modification, respectively. In the proposed project, the function of these proteins will be further characterized, both by studying recombinant proteins, as well as by phenotypic characterisation of mutant plants lacking or overexpressing these enzymes. Furthermore, some RNA viruses infecting plants have an AlkB proteins, and we have obtained results in dicating that these proteins are RNA repair enzymes involved in repairing the viral genome. As part of the proposed project, these viral AlkB proteins will be further studied, aiming to establish in more detail their role during viral infection.