Human AlkB Homolog 1 (ALKBH1) Contains Distinct AP Lyase and 6mA Demethylase Active Sites
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Bacterial AlkB and its human homologues are members of the Fe(II)/2-oxoglutarate dependent oxygenases that are known to be involved in DNA repair. AlkB homolog 1 (ALKBH1) directly repairs alkylation damage, acting as a demethylase at 3-methylcytosine (3mC) sites in single-stranded DNA. In addition to DNA repair, ALKBH1 has been shown in previous studies to have potential roles in stem cell differentiation, embryonic development, and codon recognition. A recent study has shown that ALKBH1 has the additional ability to demethylate at 6mA sites in mouse embryonic stem cells. This present study confirmed 6mA demethylase activity in Escherichia coli-produced ALKBH1 through activity assays analyzed via gel electrophoresis. This activity was shown to exist at low levels, and attempts to increase demethylation through assay variations were unsuccessful. ALKBH1 also possesses apurinic/apyrimidinic (AP) lyase activity, and this study compared both the demethylase and AP lyase activities of variants to assess the possibility of distinct active sites for these two functions. In contrast to previous studies, variant K133A did not show a significant decrease in either activity. Four variants (H113A, C118A, C129A, H134A) that were thought to be involved in a zinc finger motif were also analyzed, and this structural feature was found to be more important to demethylase activity than AP lyase activity. This finding gives evidence for distinct active sites, and may suggest a different mode of recognition for methyl and AP lesions. Comparing the two distinct functions of ALKBH1 may give further insight into its biological significance, and have important implications for DNA repair and expression.