Study for Reaction Mechanism of Damaged DNA repair enzymes using Chemically Modified DNA

建立了一条新的含2 ′-脱氧尿嘧啶核苷的寡脱氧核苷酸合成路线。在六甲基二硅氮烷存在下，保护胸苷加热得到糖基中间体，产率较高。糖基中间体与5-碘尿嘧啶在钯催化下偶联，然后对新生成的3 ′-酮基进行立体控制还原，产率较高。根据合成未修饰DNA的亚磷酰胺化学方法，将2 ′-脱氧尿嘧啶核苷（2 ′-deoxypropylduridine，2 ′-deoxypropyl Coli JM 109中进行PCR扩增，并将该基因与His/tag序列连接，构建成高效表达载体pET 16 b。通过两次柱层析得到高纯度的尿嘧啶-DNA糖基化酶，用酶活性抑制法研究了尿嘧啶-DNA糖基化酶与含2 ′-脱氧尿嘧啶核苷的寡脱氧核苷酸的相互作用。将尿嘧啶-DNA糖基化酶的底物，含2 ′-脱氧尿苷的寡核苷酸与酶在含2 ′-脱氧尿苷的寡核苷酸存在和不存在的情况下孵育。含2 ′-脱氧尿嘧啶核苷的寡核苷酸存在时的反应速率明显小于不含寡核苷酸类似物时的反应速率。结果表明，含2 ′-脱氧尿嘧啶核苷的寡核苷酸抑制尿嘧啶-DNA糖基化酶活性。

A novel practical synthetic route for oligodeoxyribonucleotides containing 2'-deoxypseudouridine has been established.A glycal intermediate was obtained by heating a protected thymidine in the presence of hexamethyldisilazane in a good yield.Palladium mediated coupling of the glycal and 5-iodouracil followed by the stereo-controlled reduction of newly generated 3'-ketone group yielded 2'-deoxypseudouridine in a good overall yield. According to the phosphoramidite chemistry generally used for synthesis of unmodified DNA, 2'-deoxypseudouridine was introduced into oligdeoxyribonucleotides.Uracil-DNA glycosylase gene (ung) was obtained from C-DNA library of E. Coli JM 109 by the PCR amplification.The high expression vector was constructed on pET16b from ung and His/tag sequence. Uracil-DNA glycosylase of high purity was obtained through two column chromatography.Interaction between uracil-DNA glycosylase and the oligodeoxyribonucleotides containing 2'-deoxypseudouridine was examined by the inhibition of the enzymatic activity. A oligonucleotide containing 2'-deoxyuridine, that is a substrate of uracil-DNA glycosylase, was incubated with the enzyme in the preence and absence of the oligonucleotide containing 2'-deoxypseudouridine. The reaction rate in the presence of the oligonucleotide containing 2'-deoxypseudouridine was obviously smaller than that in the absence of the oligonucleotide analogue. The result indicated that the the oligonucleotide containing 2'-deoxypseudouridine inhibited uracil-DNA glycosylase activity.