Bioinformatics and functional analysis define four distinct groups of AlkB DNA-dioxygenases in bacteria.

The iron(II)- and 2-oxoglutarate(2OG)-dependent dioxygenase AlkBfrom 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 (eA). EcAlkB is the only bacterial AlkBprotein characterized to date, and we here present an extensive bioinformatics and functional analysis of bacterial AlkBproteins. 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 AlkBproteins from the four different groups across the bacterial kingdom indicates a substantial degree of horizontal transfer of AlkBgenes. DNA repairactivity was associated with all tested recombinant AlkBproteins. Notably, both a group 2B protein from Xanthomonas campestrisand 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 AlkBproteins are DNA repairenzymes, and that some of these proteins do not primarily target methylated bases.