Dizdaroglu M, Zastawny T H, Carmical J R, Lloyd R S
Chemical Science and Technology Laboratory, National Institute of Standards and Technology, Gaithersburg, MD 20899, USA.
Mutat Res. 1996 Jan 2;362(1):1-8. doi: 10.1016/0921-8777(95)00025-9.
We report on a novel activity of T4 endonuclease V. This enzyme is well known to be specific for the excision of pyrimidine dimers from UV-irradiated DNA. In this work, we show that T4 endonuclease V excises 4,6-diamino-5-formamidopyrimidine from DNA. 4,6-Diamino-5-formamidopyrimidine is formed as a product of adenine in DNA upon action of hydroxyl radicals and upon UV-irradiation. DNA substrates were prepared by UV-or gamma-irradiation of DNA in aqueous solution. DNA substrates were incubated either with active T4 endonuclease V or with heat-inactivated T4 endonuclease V or without the enzyme. After incubation, DNA was precipitated and supernatant fractions were separated. Supernatant fractions after derivatization, and pellets after hydrolysis and derivatization were analyzed by gas chromatography/isotope-dilution mass spectrometry. The results provide evidence for the excision of 4,6-diamino-5-formamidopyrimidine by T4 endonuclease V from both gamma-and UV-irradiated DNA. Kinetics of excision were also determined. Fifteen other pyrimidine- and purine-derived base lesions that were identified in DNA samples were not substrates for this enzyme. It was concluded that, in addition to its well known activity for pyrimidine photodimers, T4 endonuclease V possesses an N-glycosylase activity for a major UV-radiation- and hydroxyl radical-induced monomeric product in DNA.
我们报道了T4内切核酸酶V的一种新活性。众所周知，这种酶对从紫外线照射的DNA中切除嘧啶二聚体具有特异性。在这项工作中，我们表明T4内切核酸酶V能从DNA中切除4,6-二氨基-5-甲酰胺基嘧啶。4,6-二氨基-5-甲酰胺基嘧啶是DNA中的腺嘌呤在羟基自由基作用和紫外线照射后形成的产物。DNA底物通过在水溶液中对DNA进行紫外线或γ射线照射来制备。将DNA底物与活性T4内切核酸酶V、热失活的T4内切核酸酶V或不与该酶一起孵育。孵育后，沉淀DNA并分离上清液部分。衍生化后的上清液部分以及水解和衍生化后的沉淀通过气相色谱/同位素稀释质谱法进行分析。结果为T4内切核酸酶V从γ射线和紫外线照射的DNA中切除4,6-二氨基-5-甲酰胺基嘧啶提供了证据。还测定了切除动力学。在DNA样品中鉴定出的其他15种嘧啶和嘌呤衍生的碱基损伤不是该酶的底物。得出的结论是，除了其众所周知的对嘧啶光二聚体的活性外，T4内切核酸酶V对DNA中一种主要的紫外线辐射和羟基自由基诱导的单体产物具有N-糖苷酶活性。
