Two human homologs of the Escherichia coli AlkB protein, denoted hABH2 and hABH3, were recently shown to directly reverse 1-methyladenine (1meA) and 3-methylcytosine (3meC) damages in DNA. We demonstrate that mice lacking functional mABH2 or mABH3 genes, or both, are viable and without overt phenotypes. Neither were histopathological changes observed in the gene-targeted mice. However, in the absence of any exogenous exposure to methylating agents, mice lacking mABH2, but not mABH3 defective mice, accumulate significant levels of 1meA in the genome, suggesting the presence of a biologically relevant endogenous source of methylating agent. Furthermore, embryonal fibroblasts from mABH2-deficient mice are unable to remove methyl methane sulfate (MMS)-induced 1meA from genomic DNA and display increased cytotoxicity after MMS exposure. In agreement with these results, we found that in vitro repair of 1meA and 3meC in double-stranded DNA by nuclear extracts depended primarily, if not solely, on mABH2. Our data suggest that mABH2 and mABH3 have different roles in the defense against alkylating agents.

AlkB homolog 1 (ALKBH1) is one of nine members of the family of mammalian AlkB homologs. Most Alkbh1−/− mice die during embryonic development, and survivors are characterized by defects in tissues originating from the ectodermal lineage. In this study, we show that deletion of Alkbh1 prolonged the expression of pluripotency markers in embryonic stem cells and delayed the induction of genes involved in early differentiation. In vitro differentiation to neural progenitor cells (NPCs) displayed an increased rate of apoptosis in the Alkbh1−/− NPCs when compared with wild-type cells. Whole-genome expression analysis and chromatin immunoprecipitation revealed that ALKBH1 regulates both directly and indirectly, a subset of genes required for neural development. Furthermore, our in vitro enzyme activity assays demonstrate that ALKBH1 is a histone dioxygenase that acts specifically on histone H2A. Mass spectrometric analysis demonstrated that histone H2A from Alkbh1−/− mice are improperly methylated. Our results suggest that ALKBH1 is involved in neural development by modifying the methylation status of histone H2A. Stem Cells 2012;30:2672–2682

Endogenous formation of the mutagenic DNA adduct 1,N 6 -ethenoadenine (EA) originates from lipid peroxidation. Elevated levels of EA in cancer-prone tissues suggest a role for this adduct in the development of some cancers. The base excision repair pathway has been considered the principal repair system for EA lesions until recently, when it was shown that the Escherichia coli AlkB dioxygenase could directly reverse the damage. We report here kinetic analysis of the recombinant human AlkB homologue 2 (hABH2), which is able to repair EA lesions in DNA. Furthermore, cation exchange chromatography of nuclear extracts from wild-type and mABH2 À/À mice indicates that mABH2 is the principal dioxygenase for EA repair in vivo. This is further substantiated by experiments showing that hABH2, but not hABH3, is able to complement the E. coli alkB mutant with respect to its defective repair of etheno adducts. We conclude that ABH2 is active in the direct reversal of EA lesions, and that ABH2, together with the alkyl-N-adenine-DNA glycosylase, which is the most effective enzyme for the repair of EA, comprise the cellular defense against EA lesions. [Cancer Res 2008;68(11):4142-9]