体内嘧啶二聚体-DNA糖基化酶活性的证明：以噬菌体T4感染的大肠杆菌作为模型系统
Demonstration of pyrimidine dimer-DNA glycosylase activity in vivo: bacteriophage T4-infected Escherichia coli as a model system.
作者信息
Radany E H, Friedberg E C
出版信息
J Virol. 1982 Jan;41(1):88-96. doi: 10.1128/JVI.41.1.88-96.1982.
Abstract
An approach to the detection of pyrimidine dimer-DNA glycosylase activity in living cells is presented. Mutants of Escherichia coli defective in uvr functions required for incision of UV-irradiated DNA were infected with phage T4 denV+ or denV- (defective in the T4 pyrimidine dimer-DNA glycosylase activity). In the former case the denV gene product catalyzed the incision of UV-irradiated host DNA, facilitating the subsequent excision of thymine-containing pyrimidine dimers. Isolation of these dimers from the acid-soluble fraction of infected cells was achieved by a multistep thin-layer chromatographic system. Exposure of the dimers to irradiation that monomerizes pyrimidine dimers (direct photoreversal) resulted in the stoichiometric formation of free thymine. Thus, in vivo incision of UV-irradiated DNA dependent on a pyrimidine dimer-DNA glycosylase can be demonstrated.
摘要
本文介绍了一种检测活细胞中嘧啶二聚体-DNA糖基化酶活性的方法。用噬菌体T4 denV+或denV-（T4嘧啶二聚体-DNA糖基化酶活性缺陷型）感染在UV照射DNA切割所需的uvr功能上有缺陷的大肠杆菌突变体。在前一种情况下，denV基因产物催化UV照射的宿主DNA的切割，促进随后含胸腺嘧啶的嘧啶二聚体的切除。通过多步薄层色谱系统从感染细胞的酸溶性部分分离出这些二聚体。将二聚体暴露于使嘧啶二聚体单体化的辐射（直接光逆转）下会导致化学计量的游离胸腺嘧啶的形成。因此，可以证明依赖嘧啶二聚体-DNA糖基化酶的UV照射DNA在体内的切割。
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