Novel AlkB Dioxygenases—Alternative Models for In Silico and In Vivo Studies

Abstract

BackgroundALKBH proteins, the homologs of Escherichia coli AlkB dioxygenase, constitute a direct, single-protein repair system, protecting cellular DNA and RNA against the cytotoxic and mutagenic activity of alkylating agents, chemicals significantly contributing to tumor formation and used in cancer therapy. In silico analysis and in vivo studies have shown the existence of AlkB homologs in almost all organisms. Nine AlkB homologs (ALKBH1–8 and FTO) have been identified in humans. High ALKBH levels have been found to encourage tumor development, questioning the use of alkylating agents in chemotherapy. The aim of this work was to assign biological significance to multiple AlkB homologs by characterizing their activity in the repair of nucleic acids in prokaryotes and their subcellular localization in eukaryotes.Methodology and FindingsBioinformatic analysis of protein sequence databases identified 1943 AlkB sequences with eight new AlkB subfamilies. Since Cyanobacteria and Arabidopsis thaliana contain multiple AlkB homologs, they were selected as model organisms for in vivo research. Using E. coli alkB − mutant and plasmids expressing cyanobacterial AlkBs, we studied the repair of methyl methanesulfonate (MMS) and chloroacetaldehyde (CAA) induced lesions in ssDNA, ssRNA, and genomic DNA. On the basis of GFP fusions, we investigated the subcellular localization of ALKBHs in A. thaliana and established its mostly nucleo-cytoplasmic distribution. Some of the ALKBH proteins were found to change their localization upon MMS treatment.ConclusionsOur in vivo studies showed highly specific activity of cyanobacterial AlkB proteins towards lesions and nucleic acid type. Subcellular localization and translocation of ALKBHs in A. thaliana indicates a possible role for these proteins in the repair of alkyl lesions. We hypothesize that the multiplicity of ALKBHs is due to their involvement in the metabolism of nucleo-protein complexes; we find their repair by ALKBH proteins to be economical and effective alternative to degradation and de novo synthesis.

Inducible repair of alkylated DNA in microorganisms

“…These dioxygenases reveal highly specific activity toward lesions and nucleic acid type. In contrast to EcAlkB, cyanobacterial AlkBs repaired lesions in ssRNA much more efficiently [68].…”

“…Paralogs of EcAlkB are widespread in the living world [68]. Furthermore, several isoforms/orthologs can be synthesized within one organism.…”

“…The nine human ALKBH homologs are not equally conserved in all higher eukaryotes. For instance, in Arabidopsis thaliana we have identified 13 AlkB homologs [68] and in the chicken genome (Gallus gallus), there are only seven: ALKBH1-5, ALKBH8 and FTO (Ensembl database, www.ensembl.org). Proteins encoded by these genes show a high level of identity (about 70%) with human homologues implying analogous functions, allowing chicken cells to be used as convenient model for studies on ALKBH proteins.…”

The Arabidopsis epitranscriptome

“…Two human enzymes of the AlkB family (nonheme Fe(II)/a-ketoglutarate (a-KG)-dependent dioxygenases), FTO and ALKBH5, have been shown to demethylate mRNA m 6 A [32,33]. Thirteen proteins of the AlkB family have been reported to be encoded in the Arabidopsis genome [34], but it is not yet known if any function to demethylate m 6 A. AlkB proteins were originally characterised as dioxygenases required for the repair of glycosidic bonds introduced into DNA as a result of exposure to alkylating agents. However, some AlkB family members have much wider roles, including demethylation of DNA by the stepwise oxidation of 5 methylcytosine [35].…”

1,N6-α-hydroxypropanoadenine, the acrolein adduct to adenine, is a substrate for AlkB dioxygenase

“…Its homologs are present in almost all organisms including human (18,19). A recent bioinformatic analysis of protein sequence databases has identified 1943 AlkB sequences with eight new AlkB subfamilies (20). AlkB-like DNA repair dioxygenases remove alkyl lesions from DNA bases via an oxidative mechanism restoring the native bases in the DNA.…”