Kim Min Young, Zhou Xinfeng, Delaney James C, Taghizadeh Koli, Dedon Peter C, Essigmann John M, Wogan Gerald N
Department of Biological Engineering, Center for Environmental Health Sciences, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, MA 02139, USA.
Chem Res Toxicol. 2007 Aug;20(8):1075-83. doi: 10.1021/tx700167v. Epub 2007 Jul 20.
2-Chloroacetaldehyde (CAA), a metabolite of the carcinogen vinyl chloride, reacts with DNA to form cyclic etheno ()-lesions. AlkB, an iron-/alpha-ketoglutarate-dependent dioxygenase, repairs 1, N (6)-ethenodeoxyadenosine (A) and 3, N (4)-ethenodeoxycytidine (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 A, C, and 1, N (2)-ethenodeoxyguanosine (1, N (2)-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.
2-氯乙醛（CAA）是致癌物氯乙烯的一种代谢产物，它与DNA反应形成环状乙烯基（ε）-损伤。AlkB是一种依赖铁/α-酮戊二酸的双加氧酶，它能在体内修复位点特异性修饰的单链病毒基因组中的1,N⁶-乙烯基脱氧腺苷（εA）和3,N⁴-乙烯基脱氧胞苷（εC），还能保护大肠杆菌基因组免受CAA的毒性影响。我们通过表征在体外受损并在AlkB功能正常（AlkB(+)）和AlkB缺陷（AlkB(-)）的大肠杆菌中复制的pSP189双链supF基因中诱导的突变频率、类型和分布，研究了AlkB作为细胞对CAA防御机制的作用。AlkB降低了诱变效力并提高了CAA损伤质粒的存活率。毒性和诱变数据以通过液相色谱-串联质谱（LC-MS/MS）和质粒切口测定法测得的ε-加合物和DNA链断裂水平为基准。CAA处理导致εA、εC和1,N²-乙烯基脱氧鸟苷（1,N²-G）呈剂量依赖性增加，链断裂和无碱基位点略有增加。在AlkB(+)和AlkB(-)细胞中复制的质粒中，突变频率均增加；然而，在最大CAA剂量下，AlkB(+)细胞中的突变频率比AlkB(-)细胞低5倍，这表明AlkB保护基因组免受CAA损伤。AlkB(-)细胞中大多数诱导突变是G:C到A:T的转换，G:C到T:A的颠换和A:T到G:C的转换较少。AlkB(+)细胞中G:C到A:T和A:T到G:C的转换比AlkB(-)细胞中少。G122、G123和G160处的突变热点在两种细胞类型中都很常见。在AlkB(-)细胞中还发现了另外三个热点（C133、T134和G159），AlkB(+)细胞中的突变频率降低且突变特征发生了变化。这些结果表明，AlkB蛋白有助于消除环外DNA碱基加合物，抑制这种损伤诱导的毒性和诱变后果，并有助于遗传稳定性。
