Bjelland S, Birkeland N K, Benneche T, Volden G, Seeberg E
Division for Environmental Toxicology, Norwegian Defense Research Establishment, Kjeller.
J Biol Chem. 1994 Dec 2;269(48):30489-95.
The alkA gene of Escherichia coli encodes a DNA glycosylase involved in base excision repair of DNA alkylation damage. In an attempt to define the reactions of the AlkA enzyme with methylated DNA, we discovered that the enzyme released substantial amounts of radioactivity from [methyl-3H]thymidine-labeled DNA even without any exposure of the DNA to methylating agents. The excised material was identified by chromatography as two different oxidized derivatives of thymine, 5-hydroxymethyluracil and 5-formyluracil. These products are formed in such DNA by one and two consecutive decays, respectively, of the tritiums of the labeled methyl group. Kinetic analysis showed that both the apparent Km and Vmax values for 5-formyluracil removal are within the same range as found for 3-methyladenine removal, suggesting that this catalytic property of AlkA is also significant under in vivo conditions. Removal of 5-hydroxymethyluracil proceeds at a rate that is 1-3 orders of magnitude slower. Since both 5-formyluracil and 5-hydroxymethyluracil are major products formed in DNA by exposure to ionizing radiation, these results implicate the alkA gene function also in the repair of oxidative DNA damage. Neither of the two other enzymes involved in the repair of oxidative DNA damage in E. coli, i.e. endonuclease III and formamidopyrimidine DNA glycosylase, has any affinity for oxidized unsaturated pyrimidines in DNA.
大肠杆菌的alkA基因编码一种参与DNA烷基化损伤碱基切除修复的DNA糖基化酶。为了确定AlkA酶与甲基化DNA的反应，我们发现即使DNA未暴露于甲基化剂，该酶也能从[甲基-3H]胸腺嘧啶标记的DNA中释放大量放射性。通过色谱法鉴定切除的物质为胸腺嘧啶的两种不同氧化衍生物，5-羟甲基尿嘧啶和5-甲酰基尿嘧啶。这些产物分别由标记甲基的氚经过一次和两次连续衰变在这种DNA中形成。动力学分析表明，去除5-甲酰基尿嘧啶的表观Km和Vmax值与去除3-甲基腺嘌呤时的范围相同，这表明AlkA的这种催化特性在体内条件下也很重要。去除5-羟甲基尿嘧啶的速率要慢1-3个数量级。由于5-甲酰基尿嘧啶和5-羟甲基尿嘧啶都是DNA暴露于电离辐射后形成的主要产物，这些结果表明alkA基因功能也参与氧化DNA损伤的修复。大肠杆菌中参与氧化DNA损伤修复的另外两种酶，即核酸内切酶III和甲酰胺嘧啶DNA糖基化酶，对DNA中的氧化不饱和嘧啶均无亲和力。
