title: Human AlkB homolog 1 is a mitochondrial protein that demethylates 3-methylcytosine in DNA and RNA.
abstract: The Escherichia coli AlkB protein and human homologs hABH2 and hABH3 are 2-oxoglutarate (2OG)/Fe(II)-dependent DNA/RNA demethylases that repair 1-methyladenine and 3-methylcytosine residues. Surprisingly, hABH1, which displays the strongest homology to AlkB, failed to show repair activity in two independent studies. Here, we show that hABH1 is a mitochondrial protein, as demonstrated using fluorescent fusion protein expression, immunocytochemistry, and Western blot analysis. A fraction is apparently nuclear and this fraction increases strongly if the fluorescent tag is placed at the N-terminal end of the protein, thus interfering with mitochondrial targeting. Molecular modeling of hABH1 based upon the sequence and known structures of AlkB and hABH3 suggested an active site almost identical to these enzymes. hABH1 decarboxylates 2OG in the absence of a prime substrate, and the activity is stimulated by methylated nucleotides. Employing three different methods we demonstrate that hABH1 demethylates 3-methylcytosine in single-stranded DNA and RNA in vitro. Site-specific mutagenesis confirmed that the putative Fe(II) and 2OG binding residues are essential for activity. In conclusion, hABH1 is a functional mitochondrial AlkB homolog that repairs 3-methylcytosine in single-stranded DNA and RNA.
authors.affiliation: Department of Cancer Research and Molecular Medicine, Norwegian University of Science and Technology, N-7489 Trondheim, Norway.
authors.forename: Marianne Pedersen
authors.initials: MP
authors.lastname: Westbye
authors.forename: Emadoldin
authors.initials: E
authors.lastname: Feyzi
authors.forename: Per Arne
authors.initials: PA
authors.lastname: Aas
authors.forename: Cathrine Broberg
authors.initials: CB
authors.lastname: Vågbø
authors.forename: Vivi Anita
authors.initials: VA
authors.lastname: Talstad
authors.forename: Bodil
authors.initials: B
authors.lastname: Kavli
authors.forename: Lars
authors.initials: L
authors.lastname: Hagen
authors.forename: Ottar
authors.initials: O
authors.lastname: Sundheim
authors.forename: Mansour
authors.initials: M
authors.lastname: Akbari
authors.forename: Nina-Beate
authors.initials: NB
authors.lastname: Liabakk
authors.forename: Geir
authors.initials: G
authors.lastname: Slupphaug
authors.forename: Marit
authors.initials: M
authors.lastname: Otterlei
authors.forename: Hans Einar
authors.initials: HE
authors.lastname: Krokan
mesh_terms: D000071499:AlkB Homolog 2, Alpha-Ketoglutarate-Dependent Dioxygenase; D000071500:AlkB Homolog 3, Alpha-Ketoglutarate-Dependent Dioxygenase; D003596:Cytosine / Q000031:analogs & derivatives* / Q000378:metabolism; D019175:DNA Methylation; D045643:DNA Repair Enzymes / Q000235:genetics / Q000378:metabolism*; D004272:DNA, Mitochondrial / Q000235:genetics / Q000378:metabolism*; D004277:DNA, Single-Stranded / Q000235:genetics / Q000378:metabolism*; D049308:Dioxygenases / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D024101:Mitochondrial Proteins / Q000235:genetics / Q000378:metabolism*; D006899:Mixed Function Oxygenases / Q000235:genetics / Q000378:metabolism; D012313:RNA / Q000235:genetics / Q000378:metabolism*; D012323:RNA Processing, Post-Transcriptional / Q000502:physiology; D000077278:RNA, Mitochondrial; D017386:Sequence Homology, Amino Acid
chemical_list: D004272:DNA, Mitochondrial; D004277:DNA, Single-Stranded; D029968:Escherichia coli Proteins; D024101:Mitochondrial Proteins; D000077278:RNA, Mitochondrial; C036386:3-methylcytosine; D012313:RNA; D003596:Cytosine; D006899:Mixed Function Oxygenases; D049308:Dioxygenases; C486439:ALKBH3 protein, human; D000071500:AlkB Homolog 3, Alpha-Ketoglutarate-Dependent Dioxygenase; C466427:AlkB protein, E coli; C486440:ALKBH2 protein, human; D000071499:AlkB Homolog 2, Alpha-Ketoglutarate-Dependent Dioxygenase; D045643:DNA Repair Enzymes