AlkB Influences the Chloroacetaldehyde-Induced Mutation Spectra and Toxicity in the pSP189 supF Shuttle Vector

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• 作者：Min Young Kim ; Xinfeng Zhou ; James C. Delaney ; Koli Taghizadeh ; Peter C. Dedon ; John M. Essigmann ; Gerald N. Wogan
• 刊名：Chemical Research in Toxicology
• 出版年：2007
• 出版时间：August 2007
• 年：2007
• 卷：20
• 期：8
• 页码：1075 - 1083
• 全文大小：435K
• 年卷期：v.20,no.8(August 2007)
• ISSN：1520-5010

文摘

2-Chloroacetaldehyde (CAA), a metabolite of the carcinogen vinyl chloride, reacts with DNA to form cyclic etheno (ε)-lesions. AlkB, an iron-/α-ketoglutarate-dependent dioxygenase, repairs 1,N⁶-ethenodeoxyadenosine (εA) and 3,N⁴-ethenodeoxycytidine (εC) in site-specifically modified single-stranded viral genomes in vivo and also protects the E. coli genome from the toxic effects of CAA. We examined the role of AlkB as a cellular defense against CAA by characterizing the frequencies, types, and distributions of mutations induced in the double-stranded supF gene of pSP189 damaged in vitro and replicated in AlkB-proficient (AlkB⁺) and AlkB-deficient (AlkB⁻) E.coli. AlkB reduced mutagenic potency and increased the survival of CAA-damaged plasmids. Toxicity and mutagenesis data were benchmarked to levels of ε-adducts and DNA strand breaks measured by LC-MS/MS and a plasmid nicking assay. CAA treatment caused dose-dependent increases in εA, εC, and 1,N²-ethenodeoxyguanosine (1,N²-εG) and small increases in strand breaks and abasic sites. Mutation frequency increased in plasmids replicated in both AlkB⁺ and AlkB⁻ cells; however, at the maximum CAA dose, the mutation frequency was 5-fold lower in AlkB⁺ than in AlkB⁻ cells, indicating that AlkB protected the genome from CAA lesions. Most induced mutations in AlkB⁻ cells were G:C to A:T transitions, with lesser numbers of G:C to T:A transversions and A:T to G:C transitions. G:C to A:T and A:T to G:C transitions were lower in AlkB⁺ cells than in AlkB⁻ cells. Mutational hotspots at G122, G123, and G160 were common to both cell types. Three additional hotspots were found in AlkB⁻ cells (C133, T134, and G159), with a decrease in mutation frequency and change in mutational signature in AlkB⁺ cells. These results suggest that the AlkB protein contributes to the elimination of exocyclic DNA base adducts, suppressing the toxic and mutagenic consequences induced by this damage and contributing to genetic stability.