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.
Highlights
Alkylating compounds are ubiquitous and modify cellular macromolecules such as DNA and RNA
Our results demonstrate that hABH1 is a mitochondrial DNA/ RNA demethylase, indicating that mammalian ABH1 may have dual roles in nuclei and mitochondria
We have found that hABH1 is a functional AlkB homolog that is apparently predominantly located in mitochondria
Summary
1-meA, 1-methyladenine; 3-meC, 3-methylcytosine; 2OG, 2-oxoglutarate; hABH, human AlkB homolog; PCNA, proliferating cell nuclear antigen; MMS, methyl methanesulfonate; DTT, dithiothreitol; MES, 4-morpholineethanesulfonic acid; HPLC, high pressure liquid chromatography; MS, mass spectrometry; EYFP, enhanced yellow fluorescent protein; me, methyl. The lack of AlkB-like activity of hABH1 would seem paradoxical, given the high similarity of these proteins [7]. Recent results from mAbh1Ϫ/Ϫ mice indicate that the murine Abh protein may have a role in development through participation in transcriptional regulation no enzymatic activity was demonstrated [16]. We show that the widely expressed hABH1 is a mitochondrial demethylase that demethylates 3-meC, but not 1-meA, in DNA and RNA. Results from a mouse model deficient in mAbh demonstrated a role of Abh in development, probably at the epigenetic level [16]. Our results demonstrate that hABH1 is a mitochondrial DNA/ RNA demethylase, indicating that mammalian ABH1 may have dual roles in nuclei and mitochondria
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