title: Biochemical Characterization of AP Lyase and m6A Demethylase Activities of Human AlkB Homologue 1 (ALKBH1).
abstract: Alkbh1 is one of nine mammalian homologues 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 of 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 those of 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 the cell, while the cellular role of the lyase activity (including its subsequent covalent attachment to DNA) remains uncertain.
authors.affiliation: Department of Microbiology and Molecular Genetics, Michigan State University , East Lansing, Michigan 48824, United States.
authors.forename: Tina A
authors.initials: TA
authors.lastname: Müller
authors.affiliation: Department of Microbiology and Molecular Genetics, Michigan State University , East Lansing, Michigan 48824, United States.
authors.forename: Michael A
authors.initials: MA
authors.lastname: Tobar
authors.affiliation: Biology Department, Kalamazoo College , Kalamazoo, Michigan 49006, United States.
authors.forename: Madison N
authors.initials: MN
authors.lastname: Perian
authors.affiliation: Department of Microbiology and Molecular Genetics, Michigan State University , East Lansing, Michigan 48824, United States.|Department of Biochemistry and Molecular Biology, Michigan State University , East Lansing, Michigan 48824, United States.
authors.forename: Robert P
authors.identifier: 0000-0002-3643-2054
authors.initials: RP
authors.lastname: Hausinger
mesh_terms: D000225:Adenine / Q000737:chemistry* / Q000378:metabolism; D000071498:AlkB Homolog 1, Histone H2a Dioxygenase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D020134:Catalytic Domain; D004247:DNA / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D018736:DNA Adducts; D057075:Enzyme Assays; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D006801:Humans; D006899:Mixed Function Oxygenases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D009841:Oligonucleotides / Q000737:chemistry / Q000378:metabolism; D010090:Oxidoreductases, O-Demethylating / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D017433:Protein Structure, Secondary; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013045:Species Specificity
chemical_list: D018736:DNA Adducts; D029968:Escherichia coli Proteins; D009841:Oligonucleotides; D011993:Recombinant Fusion Proteins; D004247:DNA; D006899:Mixed Function Oxygenases; D010090:Oxidoreductases, O-Demethylating; C466427:AlkB protein, E coli; C499236:ALKBH1 protein, human; D000071498:AlkB Homolog 1, Histone H2a Dioxygenase; D000225:Adenine