Identification and characterization of an intermediate in the alkali degradation of (6-4) photoproduct-containing DNA
Identification and characterization of an intermediate in the alkali degradation of (6-4) photoproduct-containing DNA
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DOI：
10.1074/jbc.m307186200
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发表时间：
2003-12-19
影响因子：
4.8
通讯作者：
Iwai, S
中科院分区：
文献类型：
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作者：
Higurashi, M;Ohtsuki, T;Iwai, S
由紫外线形成的(6-4)光产物被称为碱不稳定的DNA损伤。当紫外线照射的DNA用热碱处理时，在(6-4)光产物处发生链断裂。我们用合成的寡核苷酸分析了该光产物在碱性条件下的降解反应。制备了一种四聚体d(GT(6-4)TC)，并用高效液相色谱法测定了它在50 mM KOH中60℃时的降解情况。反应后检测到一个与起始物质的紫外吸收光谱相似的单峰，该化合物被认为是链断裂前的中间体。该中间体的形成与其他碱不稳定损伤的降解中间体进行了比较，如碱性部位、胸腺嘧啶二醇和5，6-二氢胸腺嘧啶。结果表明，(6-4)光产物碱降解的第一步是5‘-嘧啶组分的N3和C4位之间的水解。核磁共振谱和质谱学分析证实了该产物的化学结构。用XPC对络合物的形成进行分析。HR23B和DNA聚合酶ETA的跨损伤合成表明，完整光产物和水解光产物的生化性质没有明显区别。
The (6-4) photoproduct formed by ultraviolet light is known as an alkali-labile DNA lesion. Strand breaks occur at (6-4) photoproducts when UV-irradiated DNA is treated with hot alkali. We have analyzed the degradation reaction of this photoproduct under alkaline conditions using synthetic oligonucleotides. A tetramer, d(GT(6-4) TC), was prepared, and its degradation in 50 mM KOH at 60 degreesC was monitored by high performance liquid chromatography. A single peak with a UV absorption spectrum similar to that of the starting material was detected after the reaction, and this compound was regarded as an intermediate before the strand break. The formation of this intermediate was compared with intermediates from the degradation of other alkali-labile lesions such as the abasic site, thymine glycol, and 5,6-dihydrothymine. The results strongly suggested that the first step of the alkali degradation of the (6- 4) photoproduct was the hydrolysis between the N3 and C4 positions of the 5'-pyrimidine component. Analyses by NMR spectroscopy and mass spectrometry supported the chemical structure of this product. Assays of the complex formation with XPC . HR23B and the translesion synthesis by DNA polymerase eta revealed that the biochemical properties are indistinguishable between the intact and hydrolyzed photoproducts.