Biochemical Characterization of AP Lyase and m⁶A Demethylase Activities of Human AlkB Homologue 1 (ALKBH1)

Abstract

Alkbh1 is one of nine mammalian homologs of Escherichia coli AlkB, a 2-oxoglutarate-dependent dioxygenase that catalyzes direct DNA repair by removing alkyl lesions from DNA. Six distinct enzymatic activities have been reported for Alkbh1, including hydroxylation of variously methylated DNA, mRNA, tRNA, or histone substrates along with the cleavage of DNA at apurinic/apyrimidinic (AP) sites followed by covalent attachment to the 5′-product. The studies described here extend the biochemical characterization for two of these enzymatic activities using human ALKBH1: the AP lyase and 6-methyl adenine DNA demethylase activities. The steady-state and single-turnover kinetic parameters for ALKBH1 cleavage of AP sites in DNA were determined and shown to be comparable to other AP lyases. The α,β-unsaturated aldehyde of the 5′-product arising from DNA cleavage reacts predominantly with C129 of ALKBH1, but secondary sites also generate covalent adducts. The 6-methyl adenine demethylase activity was examined with a newly developed assay using a methylation-sensitive restriction endonuclease, and the enzymatic rate was found to be very low. Indeed, the demethylase activity was less than half that of the AP lyase activity when ALKBH1 samples were assayed using identical buffer conditions. The two enzymatic activities were examined using a series of site-directed variant proteins, revealing the presence of distinct, but partially overlapping active sites for the two reactions. We postulate that the very low 6-methyl adenine oxygenase activity associated with ALKBH1 is unlikely to represent the major function of the enzyme in this cell, while the cellular role of the lyase activity (including its subsequent covalent attachment to DNA) remains uncertain.

Characterization of human AlkB homolog 1 produced in mammalian cells and demonstration of mitochondrial dysfunction in ALKBH1-deficient cells

“…We previously showed that His-tagged ALKBH1 Ec possessed low m 6 A demethylase activity using ssDNA and cleaved 3′ of abasic sites in ssDNA and double-stranded (ds) DNA via β-elimination [13–15]. Here, we tested whether ALKBH1 293 also exhibits these activities.…”

“…Unusual for an AP lyase, ALKBH1 Ec is a single-turnover enzyme that forms an adduct with the α,β-unsaturated aldehyde 5′ product of the β-elimination reaction [14, 15, 20]. We tested whether ALKBH1 293 catalyzes the same covalent attachment reaction.…”

“…The m 6 A demethylase, AP lyase, and adduct formation assays were performed as previously described [14, 15, 20]. For m 6 A demethylase assays, MBP-ALKBH1 293 (5 μM) was incubated with 5′-FAM-labeled oligo_m6A1 (1 μM, 5′-AACTTCGTGCAGGCATGG G(m 6 A)TC T-TGTCTACT-3′, Sigma-Aldrich), 1 mM Fe(NH 4 ) 2 SO 4 , 1 mM L-ascorbic acid, and 1 mM 2OG in 50 mM bis-Tris (pH 7) at 37 °C for 1 h, annealed to 1.1 equivalents of oligo_m6A2 (5′-AGTAGACAA GATC CCATGCCTGCACGAAGTT-3′), and digested with DpnII (0.5 U/μl) for 1 h at 37 °C.…”

“…The protein also possesses apurinic/apyrimidinic (AP) lyase activity, cleaving DNA at abasic sites by a β-elimination mechanism. Surprisingly, ALKBH1 is a single-turnover lyase because it forms a covalent adduct with the 5′-product [13–15]. Lastly, ALKBH1 was reported to be a histone H2A dioxygenase involved in neural development [16].…”

N 6-Hydroxymethyladenine: a hydroxylation derivative of N6-methyladenine in genomic DNA of mammals

“…Recent studies also showed that the proteins of the AlkB family, such as NMAD-1 in C. elegans (23), DMAD in D. melanogaster (24), and ALKBH1 in mouse and human (27,35), can regulate m 6 dA level in genomic DNA. The in-vitro assay showed that ALKBH1 can induce the decline of m 6 dA in DNA (36,37). Along this line, we hypothesize that N 6 -hydroxymethyl-2′-deoxyadenosine (hm 6 dA) can be formed from the hydroxylation of m 6 dA by ALKBH1 in genomic DNA of mammals (Figure 1).…”