Division of Chemistry, Graduate School of Engineering Science, Osaka University, 1-3 Machikaneyama, Toyonaka, Osaka 560-8531, Japan.
Org Biomol Chem. 2013 Jun 7;11(21):3526-34. doi: 10.1039/c3ob00012e. Epub 2013 Apr 17.
The (6-4) photoproduct is one of the major UV-induced lesions in DNA. We previously showed that hydrolytic ring opening of the 5' base and subsequent hydrolysis of the glycosidic bond of the 3' component occurred when this photoproduct was treated with aqueous NaOH. In this study, we found that another product was obtained when the (6-4) photoproduct was heated at 90 °C for 6 h, in a 0.1 M solution of N,N'-dimethyl-1,2-ethanediamine adjusted to pH 7.4 with acetic acid. An analysis of the chemical structure of this product revealed that the 5' base was intact, whereas the glycosidic bond at the 3' component was hydrolyzed in the same manner. The strand break was detected for a 30-mer oligonucleotide containing the (6-4) photoproduct upon treatment with the above solution or other pH 7.4 solutions containing biogenic amines, such as spermidine and spermine. In the case of spermidine, the rate constant was calculated to be 1.4 × 10(-8) s(-1) at 37 °C. The strand break occurred even when the oligonucleotide was heated at 90 °C in 0.1 M sodium phosphate (pH 7.0), although this treatment produced several types of 5' fragments. The Dewar valence isomer was inert to this reaction. The product obtained from the (6-4) photoproduct-containing 30-mer was used to investigate the enzymatic processing of the 3' end bearing the damaged base and a phosphate. The ERCC1-XPF complex removed several nucleotides containing the damaged base, in the presence of replication protein A.
（6-4）光产物是 DNA 中主要的紫外线诱导损伤之一。我们之前曾表明，当用氢氧化钠水溶液处理该光产物时，5'碱基的水解环打开，随后 3'组分的糖苷键水解。在这项研究中，我们发现当（6-4）光产物在 90°C 下加热 6 小时时，在 pH 为 7.4 的 0.1M N,N'-二甲-1,2-乙二胺溶液中，会得到另一种产物。该产物的化学结构分析表明，5'碱基完好无损，而 3'组分的糖苷键以相同的方式水解。当用上述溶液或其他含有生物胺（如亚精胺和精胺）的 pH 为 7.4 的溶液处理含有（6-4）光产物的 30 聚核苷酸时，检测到该链断裂。在亚精胺的情况下，在 37°C 下的速率常数计算为 1.4×10(-8) s(-1)。即使在 0.1M 磷酸钠（pH 7.0）中于 90°C 加热寡核苷酸时，也会发生链断裂，尽管这种处理会产生几种类型的 5'片段。Dewar 价态异构体对此反应无活性。从含有（6-4）光产物的 30 聚核苷酸获得的产物用于研究带有损伤碱基和磷酸的 3'末端的酶处理。在复制蛋白 A 的存在下，ERCC1-XPF 复合物去除了几个含有损伤碱基的核苷酸。
