Berdal K G, Bjørås M, Bjelland S, Seeberg E
Norwegian Defence Research Establishment, Division for Environmental Toxicology, Kjeller, Norway.
EMBO J. 1990 Dec;9(13):4563-8. doi: 10.1002/j.1460-2075.1990.tb07909.x.
An alkylation repair deficient mutant of Escherichia coli (tag ada), lacking DNA glycosylase activity for removal of alkylated bases, was transformed by a genomic yeast DNA library and clones selected which survived plating on medium containing the alkylating agent methylmethane sulphonate. Three distinct yeast clones were identified which were able to suppress the alkylation sensitive phenotype of the bacterial mutant. Restriction enzyme analysis revealed common DNA fragments present in all three clones spanning 2 kb of yeast DNA. DNA from this region was sequenced and analysed for possible translation of polypeptides with any homology to either the Tag or the AlkA DNA glycosylases of E. coli. One open reading frame of 296 amino acids was identified encoding a putative protein with significant homology to AlkA. DNA containing the open reading frame was subcloned in E. coli expression vectors and cell extracts assayed for alkylbase DNA glycosylase activity. It appeared that such activity was expressed at levels sufficiently high for enzyme purification. The molecular weight of the purified protein was determined by SDS-PAGE to be 35,000 daltons, in good agreement with the 34,340 value calculated from the sequence. The yeast enzyme was able to excise 7-methylguanine as well as 3-methyladenine from dimethyl sulphate treated DNA, confirming the related nature of this enzyme to the AlkA DNA glycosylase from E. coli.
缺乏用于去除烷基化碱基的DNA糖基化酶活性的大肠杆菌烷基化修复缺陷型突变体（tag ada），用基因组酵母DNA文库进行转化，并筛选出在含有烷基化剂甲磺酸甲酯的培养基上平板培养后存活的克隆。鉴定出三个不同的酵母克隆，它们能够抑制细菌突变体的烷基化敏感表型。限制性内切酶分析显示，所有三个克隆中都存在跨越2 kb酵母DNA的共同DNA片段。对该区域的DNA进行测序，并分析是否可能翻译出与大肠杆菌的Tag或AlkA DNA糖基化酶具有任何同源性的多肽。鉴定出一个由296个氨基酸组成的开放阅读框，编码一种与AlkA具有显著同源性的推定蛋白质。将含有开放阅读框的DNA亚克隆到大肠杆菌表达载体中，并对细胞提取物进行烷基碱基DNA糖基化酶活性检测。结果表明，这种活性的表达水平足以进行酶的纯化。通过SDS-PAGE测定纯化蛋白的分子量为35,000道尔顿，与根据序列计算出的34,340的值非常一致。酵母酶能够从硫酸二甲酯处理的DNA中切除7-甲基鸟嘌呤以及3-甲基腺嘌呤，证实了该酶与大肠杆菌的AlkA DNA糖基化酶的相关性。
