AlkB influences the chloroacetaldehyde-induced mutation spectra and toxicity in the pSP189 supF shuttle vector
AlkB influences the chloroacetaldehyde-induced mutation spectra and toxicity in the pSP189 supF shuttle vector
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DOI：
10.1021/tx700167v
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发表时间：
2007-08-01
影响因子：
4.1
通讯作者：
Wogan, Gerald N.
中科院分区：
文献类型：
--
作者：
Kim, Min Young;Zhou, Xinfeng;Wogan, Gerald N.
2-氯乙醛(CAA)是致癌物氯乙烯的代谢物，与DNA反应形成环状亚乙基(Epsilon)损伤。AlkB是一种依赖铁/α-酮戊二酸的双加氧酶，在体内修复定点修饰的单链病毒基因组中的1，N-6-亚乙基脱氧腺苷(Epsilon A)和3，N-4-亚乙基脱氧胞苷(Epsilon C)，并保护大肠杆菌基因组免受CAA的毒性影响。我们通过鉴定在体外损伤的pSP189双链supF基因的突变频率、类型和分布，以及在AlkB熟练(AlkB(+))和AlkB缺乏(AlkB(-))的大肠杆菌中复制的突变，来研究AlkB作为细胞防御CAA的作用。AlkB降低了CAA损伤的质粒的诱变能力，提高了其存活率。毒性和致突变数据以LC-MS/MS和质粒划痕试验测量的-加合物和DNA链断裂水平为基准。CAA处理后，epsilon A、epsilon C和1，N-2-亚乙基脱氧鸟苷(1，N-2-epsilon G)的含量呈剂量依赖性增加，链断裂和基本部位略有增加。在AlkB(+)和AlkB(-)细胞中复制的质粒的突变频率增加；然而，在最大CAA剂量下，AlkB(+)细胞的突变频率比AlkB(-)细胞低5倍，这表明AlkB保护基因组免受CAA损伤。AlkB(-)细胞的突变多为G：C到A：T转换，少数为G：C到T：A转换和A：T到G：C转换。在AlkB(+)细胞中，G：C到A：T和A：T到G：C的转变低于AlkB细胞。位于G122、G123和G160的突变热点在两种类型的细胞中都是共同的。在AlkB(-)细胞(C133、T134和G159)中发现了另外3个热点，AlkB(+)细胞的突变频率和突变特征发生了变化。这些结果表明，AlkB蛋白有助于消除外环DNA碱基加合物，抑制这种损伤引起的毒性和突变后果，并有助于遗传稳定。
2-Chloroacetaldehyde (CAA), a metabolite of the carcinogen vinyl chloride, reacts with DNA to form cyclic etheno (epsilon)-lesions. AlkB, an iron-/alpha-ketoglutarate-dependent dioxygenase, repairs 1, N-6-ethenodeoxyadenosine (epsilon A) and 3, N-4-ethenodeoxycytidine (epsilon C) 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 -adducts and DNA strand breaks measured by LC-MS/MS and a plasmid nicking assay. CAA treatment caused dose-dependent increases in epsilon A, epsilon C, and 1, N-2-ethenodeoxyguanosine (1, N-2-epsilon G) 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.