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1.  AlkB homolog 2 (ABH2) mediated repair of ethenoadenine lesions in mammalian DNA 
Cancer research  2008;68(11):4142-4149.
Endogenous formation of the mutagenic DNA adduct 1,N6-ethenoadenosine (εA) originates from lipid peroxidation. Elevated levels of εA in cancer-prone tissues suggest a role for this adduct in the development of some cancers. The base excision repair (BER) pathway has been considered the principal repair system for εA lesions until recently, when it was shown that the Escherichia coli (E. coli) AlkB dioxygenase could directly reverse the damage. We report here kinetic analysis of the recombinant human AlkB homolog 2 (hABH2), which is able to repair εA lesions in DNA. Furthermore, cation exchange chromatography of nuclear extracts from wild-type and mABH2−/− mice indicate that mABH2 is the principal dioxygenase for εA 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 direct reversal of εA lesions and that ABH2 together with the ANPG glycosylase, which is the most effective enzyme for repair of εA, comprise the cellular defense against εA lesions.
PMCID: PMC4725713  PMID: 18519673
DNA repair; ABH2; AlkB; ethenoadenine
2.  Bioinformatics and functional analysis define four distinct groups of AlkB DNA-dioxygenases in bacteria 
Nucleic Acids Research  2009;37(21):7124-7136.
The iron(II)- and 2-oxoglutarate (2OG)-dependent dioxygenase AlkB from Escherichia coli (EcAlkB) repairs alkylation damage in DNA by direct reversal. EcAlkB substrates include methylated bases, such as 1-methyladenine (m1A) and 3-methylcytosine (m3C), as well as certain bulkier lesions, for example the exocyclic adduct 1,N6-ethenoadenine (εA). EcAlkB is the only bacterial AlkB protein characterized to date, and we here present an extensive bioinformatics and functional analysis of bacterial AlkB proteins. Based on sequence phylogeny, we show that these proteins can be subdivided into four groups: denoted 1A, 1B, 2A and 2B; each characterized by the presence of specific conserved amino acid residues in the putative nucleotide-recognizing domain. A scattered distribution of AlkB proteins from the four different groups across the bacterial kingdom indicates a substantial degree of horizontal transfer of AlkB genes. DNA repair activity was associated with all tested recombinant AlkB proteins. Notably, both a group 2B protein from Xanthomonas campestris and a group 2A protein from Rhizobium etli repaired etheno adducts, but had negligible activity on methylated bases. Our data indicate that the majority, if not all, of the bacterial AlkB proteins are DNA repair enzymes, and that some of these proteins do not primarily target methylated bases.
PMCID: PMC2790896  PMID: 19786499
3.  Viral AlkB proteins repair RNA damage by oxidative demethylation 
Nucleic Acids Research  2008;36(17):5451-5461.
Bacterial and mammalian AlkB proteins are iron(II)- and 2-oxoglutarate-dependent dioxygenases that reverse methylation damage, such as 1-methyladenine and 3-methylcytosine, in RNA and DNA. An AlkB-domain is encoded by the genome of numerous single-stranded, plant-infecting RNA viruses, the majority of which belong to the Flexiviridae family. Our phylogenetic analysis of AlkB sequences suggests that a single plant virus might have acquired AlkB relatively recently, followed by horizontal dissemination among other viruses via recombination. Here, we describe the first functional characterization of AlkB proteins from three plant viruses. The viral AlkB proteins efficiently reactivated methylated bacteriophage genomes when expressed in Escherichia coli, and also displayed robust, iron(II)- and 2-oxoglutarate-dependent demethylase activity in vitro. Viral AlkB proteins preferred RNA over DNA substrates, and thus represent the first AlkBs with such substrate specificity. Our results suggest a role for viral AlkBs in maintaining the integrity of the viral RNA genome through repair of deleterious methylation damage, and support the notion that AlkB-mediated RNA repair is biologically relevant.
PMCID: PMC2553587  PMID: 18718927

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