Characterization of human AlkB homolog 1 produced in mammalian cells and demonstration of mitochondrial dysfunction in ALKBH1-deficient cells
- Microbiology and Molecular Genetics, Michigan State University, East Lansing, MI 48824 (United States)
Highlights: • Mammalian produced ALKBH1 (ALKBH1{sub 293}) possesses m{sup 6}A demethylase and AP lyase activity. • ALKBH1{sub 293} forms an adduct with the 5′-product of the AP lyase reaction. • ALKBH1 localizes to the mitochondria in MSU1.1 and HEK293 cells. • ALKBH1-deficient cells exhibit mitochondrial dysfunction. Alkbh1 is a mammalian homolog of the Escherichia coli DNA repair enzyme AlkB, an Fe(II) and 2-oxoglutarate dependent dioxygenase that removes alkyl lesions from DNA bases. The human homolog ALKBH1 has been associated with six different enzymatic activities including DNA, mRNA, or tRNA hydroxylation, cleavage at abasic (AP) sites in DNA, as well as demethylation of histones. The reported cellular roles of this protein reflect the diverse enzymatic activities and include direct DNA repair, tRNA modification, and histone modification. We demonstrate that ALKBH1 produced in mammalian cells (ALKBH1{sub 293}) is similar to the protein produced in bacteria (ALKBH1{sub Ec}) with regard to its m{sup 6}A demethylase and AP lyase activities. In addition, we find that ALKBH1{sub 293} forms a covalent adduct with the 5′ product of the lyase product in a manner analogous to ALKBH1{sub Ec}. Localization and subcellular fractionation studies with the endogenous protein in two human cell strains confirm that ALKBH1 is primarily in the mitochondria. Two strains of CRISPR/Cas9-created ALKBH1-deficient HEK293 cells showed increases in mtDNA copy number and mitochondrial dysfunction as revealed by growth measurements and citrate synthase activity assays.
- OSTI ID:
- 23100650
- Journal Information:
- Biochemical and Biophysical Research Communications, Journal Name: Biochemical and Biophysical Research Communications Journal Issue: 1 Vol. 495; ISSN 0006-291X; ISSN BBRCA9
- Country of Publication:
- United States
- Language:
- English
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