Spectroscopic analysis of the pyrimidine(6-4)pyrimidone photoproduct: insights into the (6-4) photolyase reaction
Spectroscopic analysis of the pyrimidine(6-4)pyrimidone photoproduct: insights into the (6-4) photolyase reaction
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DOI：
10.1039/b815458a
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发表时间：
2009-01-01
影响因子：
3.2
通讯作者：
Iwai, Shigenori
中科院分区：
文献类型：
--
作者：
Yamamoto, Junpei;Tanaka, Yoshiyuki;Iwai, Shigenori
我们合成了N-15标记的（6-4）光产物的二核苷单磷酸，这是DNA中主要的紫外线诱导损伤之一，为了研究（6-4）光解酶的修复机制，并通过测量N-15核磁共振光谱来表征其质子化状态，并通过pH值的函数来表征它。我们期望在pH值为3时，嘧啶（15）N3由于质子化而发生化学位移变化，就像N-15标记的5-甲基嘧啶-2- 1核苷一样。然而，有趣的是，这种变化只在碱性溶液中观察到。在酸性条件下的紫外吸收光谱和高效液相色谱分析中，在pH值为1及以下时观察到质子化诱导水解的最大吸收波长的变化，而在pH值为2和3之间时，5-甲基嘧啶-2- 1的质子化发生了变化。这些结果表明，该N3的pK(a)值明显低于正常的嘧啶环，并强烈提示在生理条件下，3‘基的N3与5’基的5- oh之间形成了分子内氢键。本研究结果不仅对（6-4）光解酶的识别和反应机制有重要意义，而且对（6-4）光产物的化学性质也有重要意义。
We synthesized a dinucleoside monophosphate of the N-15-labeled (6-4) photoproduct, which is one of the major UV-induced lesions in DNA, to investigate the (6-4) photolyase repair mechanism, and characterized its protonation state by measuring N-15 NMR spectra as a function of pH. We expected that chemical-shift changes of the pyrimidone (15)N3, due to protonation, would be observed at pH 3, as observed for the N-15-labeled 5-methylpyrimidin-2-one nucleoside. Interestingly, however, the changes were observed only in alkaline solutions. In UV absorption spectroscopy and HPLC analyses under acidic conditions, a change in the maximum absorption wavelength, due to the protonation-induced hydrolysis, was observed at and below pH 1, but not at pH 2, whereas the protonation of 5-methylpyrimidin-2-one occurred at pH values between 2 and 3. These results indicated that the pK(a) value for this N3 is remarkably lower than that of a normal pyrimidone ring, and strongly suggest that an intramolecular hydrogen bond is formed between the N3 of the 3' base and the 5-OH of the 5' base under physiological conditions. The results of this study have implications not only for the recognition and reaction mechanisms of (6-4) photolyase, but also for the chemical nature of the (6-4) photoproduct.