Sassanfar M, Dosanjh M K, Essigmann J M, Samson L
Laboratory of Toxicology, Harvard School of Public Health, Boston, Massachusetts 02115.
J Biol Chem. 1991 Feb 15;266(5):2767-71.
The suicidal inactivation mechanism of DNA repair methyltransferases (MTases) was exploited to measure the relative efficiencies with which the Escherichia coli, human, and Saccharomyces cerevisiae DNA MTases repair O6-methylguanine (O6MeG) and O4-methylthymine (O4MeT), two of the DNA lesions produced by mutagenic and carcinogenic alkylating agents. Using chemically synthesized double-stranded 25-base pair oligodeoxynucleotides containing a single O6MeG or a single O4MeT, the concentration of O6MeG or O4MeT substrate that produced 50% inactivation (IC50) was determined for each of four MTases. The E. coli ogt gene product had a relatively high affinity for the O6MeG substrate (IC50 8.1 nM) but had an even higher affinity for the O4MeT substrate (IC50 3 nM). By contrast, the E. coli Ada MTase displayed a striking preference for O6MeG (IC50 1.25 nM) as compared to O4MeT (IC50 27.5 nM). Both the human and the yeast DNA MTases were efficiently inactivated upon incubation with the O6MeG-containing oligomer (IC50 values of 1.5 and 1.3 nM, respectively). Surprisingly, the human and yeast MTases were also inactivated by the O4MeT-containing oligomer albeit at IC50 values of 29.5 and 44 nM, respectively. This result suggests that O4MeT lesions can be recognized in this substrate by eukaryotic DNA MTases but the exact biochemical mechanism of methyltransferase inactivation remains to be determined.
DNA修复甲基转移酶（MTases）的自杀性失活机制被用于测定大肠杆菌、人类和酿酒酵母DNA MTases修复O6-甲基鸟嘌呤（O6MeG）和O4-甲基胸腺嘧啶（O4MeT）的相对效率，这两种DNA损伤是由诱变和致癌性烷基化剂产生的。使用化学合成的含有单个O6MeG或单个O4MeT的25碱基对双链寡脱氧核苷酸，测定了四种MTases中每种酶使50%失活（IC50）的O6MeG或O4MeT底物浓度。大肠杆菌ogt基因产物对O6MeG底物具有相对较高的亲和力（IC50为8.1 nM），但对O4MeT底物具有更高的亲和力（IC50为3 nM）。相比之下，大肠杆菌Ada MTase对O6MeG（IC50为1.25 nM）的偏好明显高于O4MeT（IC50为27.5 nM）。人类和酵母DNA MTases与含O6MeG的寡聚物孵育后均有效失活（IC50值分别为1.5和1.3 nM）。令人惊讶的是，人类和酵母MTases也被含O4MeT的寡聚物失活，尽管IC50值分别为29.5和44 nM。这一结果表明，真核DNA MTases可以在该底物中识别O4MeT损伤，但甲基转移酶失活的确切生化机制仍有待确定。
