Identification and characterisation of the Drosophila melanogaster O6-alkylguanine-DNA alkyltransferase cDNA
Identification and characterisation of the Drosophila melanogaster O6-alkylguanine-DNA alkyltransferase cDNA
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DOI：
10.1093/nar/27.8.1795
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发表时间：
1999-04-15
影响因子：
14.9
通讯作者：
Pastink, A
中科院分区：
文献类型：
--
作者：
Kooistra, R;Zonneveld, JBM;Pastink, A
蛋白质O-6-烷基鸟嘌呤-DNA烷基转移酶（烷基转移酶）参与DNA中O-6-烷基鸟嘌呤和O-4-烷基胸腺嘧啶的修复，在大多数生物体中在减弱某些类别烷化剂的细胞毒性和致突变作用方面发挥重要作用。根据与酿酒酵母和人的相应基因的序列同源性，鉴定了一个包含果蝇烷基转移酶基因（DmAGT）的基因组克隆，DmAGT基因位于第三染色体上的84 A位。DmAGT cDNA的核苷酸序列分析表明，DmAGT cDNA的开放阅读框编码194个氨基酸。通过表达DmAGT cDNA拯救了烷基转移酶缺陷型细菌的MNNG超敏表型。此外，烷基转移酶的活性被确定在大肠杆菌的粗提取物窝藏DmAGT cDNA和这种活性被抑制预孵育的提取物含有一个单一的O-6-甲基鸟嘌呤病变的寡核苷酸。与大肠杆菌Ogt和酵母烷基转移酶相似，但与人类烷基转移酶相反，果蝇烷基转移酶对O-6-苄基鸟嘌呤的失活具有抗性。在大肠杆菌lacZ回复突变试验中，DmAGT的表达在体内有效地抑制了MNNG诱导的G：C-->A：T以及A：T-->G：C转换突变。这些结果表明，存在的烷基转移酶特异性修复O-6-甲基鸟嘌呤和O-4-甲基胸腺嘧啶在果蝇。
The protein O-6-alkylguanine-DNA alkyltransferase (alkyltransferase) is involved in the repair of O-6-alkylguanine and O-4-alkylthymine in DNA and plays an important role in most organisms in attenuating the cytotoxic and mutagenic effects of certain classes of alkylating agents. A genomic clone encompassing the Drosophila melanogaster alkyltransferase gene (DmAGT) was identified on the basis of sequence homology with corresponding genes in Saccharomyces cerevisiae and man. The DmAGT gene is located at position 84A on the third chromosome. The nucleotide sequence of DmAGT cDNA revealed an open reading frame encoding 194 amino acids. The MNNG-hypersensitive phenotype of alkyltransferase-deficient bacteria was rescued by expression of the DmAGT cDNA. Furthermore, alkyltransferase activity was identified in crude extracts of Escherichia coli harbouring DmAGT cDNA and this activity was inhibited by preincubation of the extract with an oligonucleotide containing a single O-6-methylguanine lesion. Similar to E.coli Ogt and yeast alkyltransferase but in contrast to the human alkyltransferase, the Drosophila alkyltransferase is resistant to inactivation by O-6-benzylguanine. In an E.coli lacZ reversion assay, expression of DmAGT efficiently suppressed MNNG-induced G:C-->A:T as well as A:T-->G:C transition mutations in vivo. These results demonstrate the presence of an alkyltransferase specific for the repair of O-6-methylguanine and O-4-methylthymine in Drosophila.