title: The role of AlkB protein in repair of 1,N⁶-ethenoadenine in Escherichia coli cells.
abstract: Etheno (ε) DNA adducts, including 1,N(6)-ethenoadenine (εA), are formed by various bifunctional agents of exogenous and endogenous origin. The AT→TA transversion, the most frequent mutation provoked by the presence of εA in DNA, is very common in critical codons of the TP53 and RAS genes in tumours induced by exposure to carcinogenic vinyl compounds. Here, using a method that allows examination of the mutagenic potency of a metabolite of vinyl chloride, chloroacetaldehyde (CAA), but eliminates its cytotoxicity, we studied the participation of alkA, alkB and mug gene products in the repair of εA in Escherichia coli cells. The test system used comprised the pIF105 plasmid bearing the lactose operon of CC105 origin, which allowed monitoring of Lac(+) revertants that arose by AT→TA substitutions due to the modification of adenine by CAA. The plasmid was CAA-modified in vitro and replicated in E.coli of various genetic backgrounds (wt, alkA, alkB, mug, alkAalkB, alkAmug and alkBmug). To modify the levels of the AlkA and AlkB proteins, mutagenesis was studied in E.coli cells induced or not in adaptive response to alkylating agents. Considering the levels of CAA-induced Lac(+) revertants in strains harbouring the CAA-modified pIF105 plasmid and induced or not in adaptive response, we conclude that the AlkB dioxygenase plays a major role in decreasing the level of AT→TA mutations, thus in the repair of εA in E.coli cells. The observed differences of mutation frequencies in the various mutant strains assayed indicate that Mug glycosylase is also engaged in the repair of εA, whereas the role the AlkA glycosylase in this repair is negligible.
authors.affiliation: Institute of Biochemistry and Biophysics, Department of Molecular Biology, Polish Academy of Sciences, 5A Pawińskiego Street, 02-106 Warsaw, Poland.
authors.forename: Agnieszka M
authors.initials: AM
authors.lastname: Maciejewska
authors.forename: Beata
authors.initials: B
authors.lastname: Sokołowska
authors.forename: Adam
authors.initials: A
authors.lastname: Nowicki
authors.forename: Jarosław T
authors.initials: JT
authors.lastname: Kuśmierek
mesh_terms: D000079:Acetaldehyde / Q000031:analogs & derivatives* / Q000378:metabolism / Q000633:toxicity; D000225:Adenine / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism; D003114:Colony-Forming Units Assay; D018736:DNA Adducts / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D045647:DNA Glycosylases / Q000235:genetics; D004260:DNA Repair / Q000235:genetics*; D018274:Electroporation; D004926:Escherichia coli; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism*; D006899:Mixed Function Oxygenases / Q000235:genetics / Q000378:metabolism*; D015394:Molecular Structure; D016296:Mutagenesis; D010957:Plasmids / Q000235:genetics; D018709:Statistics, Nonparametric; D044502:Thymine DNA Glycosylase / Q000235:genetics / Q000378:metabolism*
chemical_list: C051141:1,N(6)-ethanoadenine; D018736:DNA Adducts; D029968:Escherichia coli Proteins; C004656:chloroacetaldehyde; D006899:Mixed Function Oxygenases; C466427:AlkB protein, E coli; D045647:DNA Glycosylases; D044502:Thymine DNA Glycosylase; C113921:mismatch-specific thymine uracil-DNA glycosylase; C076131:DNA-3-methyladenine glycosidase II; D000079:Acetaldehyde; D000225:Adenine