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

Abstract

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.

Rhein Inhibits AlkB Repair Enzymes and Sensitizes Cells to Methylated DNA Damage

“…Direct-acting chemicals constantly damage nucleic acids and generate various methyl lesions with mutagenic and/or cytotoxic consequences (2,3). O 6 -Methylguanine (O 6 mG) 2 and N 3 -methyladenine lesions have the highest potential for methylating damage by an SN1 agent such as N-methyl-NЈ-nitro-N-nitrosoguanidine (MNNG), which block replication and are thought to be toxic (4,5). For the most part, the SN2 agent such as methyl methane sulfonate (MMS) produces N 1 -methyladenine (m 1 A) and N 3 -methylcytosine (m 3 C) lesions in single-stranded DNA (ssDNA).…”

“…For the most part, the SN2 agent such as methyl methane sulfonate (MMS) produces N 1 -methyladenine (m 1 A) and N 3 -methylcytosine (m 3 C) lesions in single-stranded DNA (ssDNA). Accumulation of these adducts can lead to cell death (6,7). Organisms have evolved several mechanisms to efficiently remove various methyl lesions, including suicidal methyltransferases, DNA glycosylases, and the AlkB family dioxygenases (see Fig.…”

Protozoan ALKBH8 Oxygenases Display both DNA Repair and tRNA Modification Activities

“…In fact, the AT protein was investigated by us previously, but we failed to purify soluble recombinant protein [38]. It is included here because it is possible to generate gene knock-outs of the corresponding bacterium by the so-called TargeTron technology [45].…”

“…Interestingly, when AlkB proteins from the four groups were investigated, the tested proteins all displayed repair activity on DNA. Notably, the tested ALKBH8-like protein, originating from Rhizobium etli ( R. etli ), did not show DNA repair activity on methylated bases, but rather on etheno adducts [38]. Plants, such as Arabidopsis thaliana , possess putative orthologues of several of the ALKBH proteins found in mammals, including ALKBH8.…”

Theoretical Investigations on Structure and Function of Human Homologue hABH4 of E.coli ALKB4

“…Bioinformatics methods had been used previously for the finding of various functions as well as to see the relatedness between the different human homologues of Alkb proteins 2 . It was also observed, in another study, that majority of the bacterial AlkB proteins are DNA repair enzymes, and some of these proteins do not primarily target methylated bases 3 .…”