Mouse models for studying oxidative demethylation of DNA and RNA

It was recently discovered that the AlkB protein from E. coli uses a novel mechanism, termed oxidative demethylation, to remove deleterious methyl lesions from DNA and RNA. Eight mammalian AlkB homologues, denoted ABH1 - ABH8 (mABH1-8 in mice), exist, and two of these, ABH2 and ABH3, have been shown to share the ability of E. coli AlkB to demethylate damaged nucleic acids. The function of the remaining homologues remains elusive; they may be repair proteins, but they may also be involved in demethylating endogenous, reversible methyl modifications in RNA or DNA. The proposed project is a collaborative effort between the groups of Pål Ø. Falnes and Arne Klungland, and is aimed at functionally characterizing mammalian AlkB proteins through studying mice wh ere the corresponding genes have been ablated or altered. The project will take advantage of the already established knock-out mice representing the functions mABH1, mABH2, and mABH3, and novel mouse models will also be developed, with main focus on the m ABH8 function. The mice will be studied at several levels; the amount of the relevant methyl adducts in mouse tissues and mouse-derived cell lines will be measured, as well as kinetics of demethylation reactions. Furthermore, novel in vitro assays will be developed for studying demethylase activities in mouse-derived cellular extracts, as well as for studying the effects of defective demethylation.