title: Methylation damage to RNA induced in vivo in Escherichia coli is repaired by endogenous AlkB as part of the adaptive response.
abstract: Cytotoxic 1-methyladenine (1-meA) and 3-methylcytosine (3-meC) lesions induced in DNA and RNA in vitro and in pre-damaged DNA and RNA bacteriophages in vivo are repaired by the Escherichia coli (E. coli) protein AlkB and a human homolog, ALKBH3. However, it is not known whether endogenous RNA is repaired in vivo by repair proteins present at physiological concentrations. The concept of RNA repair as a biologically relevant process has therefore remained elusive. Here, we demonstrate AlkB-mediated repair of endogenous RNA in vivo by measuring differences in lesion-accumulation in two independent AlkB-proficient and deficient E. coli strains during exposure to methyl methanesulfonate (MMS). Repair was observed both in AlkB-overproducing strains and in the wild-type strains after AlkB induction. RNA repair appeared to be highest in RNA species below 200 nucleotides in size, mainly comprising tRNAs. Strikingly, at least 10-fold more lesions were repaired in RNA than in DNA. This may be a consequence of some 30-fold higher levels of aberrant methylation in RNA than in DNA after exposure to MMS. A high primary kinetic isotope effect (>10) was measured using a deuterated methylated RNA substrate, D3-1me(rA), demonstrating that it is the catalytic step, and not the search step that is rate-limiting. Our results demonstrate that RNA repair by AlkB takes place in endogenous RNA as part of an adaptive response in wild-type E. coli cells.
authors.affiliation: Department of Cancer Research and Molecular Medicine, Faculty of Medicine, Norwegian University of Science and Technology, Trondheim, Norway.
authors.forename: Cathrine Broberg
authors.initials: CB
authors.lastname: Vågbø
authors.forename: Eva K
authors.initials: EK
authors.lastname: Svaasand
authors.forename: Per A
authors.initials: PA
authors.lastname: Aas
authors.forename: Hans E
authors.initials: HE
authors.lastname: Krokan
mesh_terms: D000222:Adaptation, Physiological / Q000235:genetics; D000477:Alkylating Agents / Q000494:pharmacology; D004260:DNA Repair; D004269:DNA, Bacterial / Q000235:genetics; D004790:Enzyme Induction; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D029968:Escherichia coli Proteins / Q000502:physiology*; D007700:Kinetics; D008741:Methyl Methanesulfonate / Q000494:pharmacology; D008745:Methylation; D006899:Mixed Function Oxygenases / Q000502:physiology*; D012329:RNA, Bacterial / Q000235:genetics* / Q000378:metabolism
chemical_list: D000477:Alkylating Agents; D004269:DNA, Bacterial; D029968:Escherichia coli Proteins; D012329:RNA, Bacterial; D008741:Methyl Methanesulfonate; D006899:Mixed Function Oxygenases; C466427:AlkB protein, E coli