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

利用化学修饰DNA研究受损DNA修复酶的反应机制

基本信息

批准号：
12680595
负责人：
ONO Akira
金额：
$ 1.73万
依托单位：
Tokyo Metropolitan University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2000
资助国家：
日本
起止时间：
2000 至 2001
项目状态：
已结题

项目摘要

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.

已经建立了包含2'-脱氧假尿苷的寡脱氧核糖核苷酸的新颖实用合成路线。通过在六甲基二硅氮烷存在下加热受保护的胸苷，获得了糖醛中间体，收率良好。钯介导的糖醛和5-碘尿嘧啶的偶联，然后进行立体反应新生成的3'-酮基的受控还原以良好的总产率产生2'-脱氧假尿苷。根据一般用于合成未修饰DNA的亚磷酰胺化学，将2'-脱氧假尿苷引入到寡脱氧核糖核苷酸中。通过PCR扩增从大肠杆菌JM 109的C-DNA文库中获得尿嘧啶-DNA糖基化酶基因(ung)。根据 ung 和 His/tag 序列在 pET16b 上构建表达载体。通过两柱层析获得了高纯度的尿嘧啶-DNA糖基化酶。通过抑制酶活性来研究尿嘧啶-DNA糖基化酶与含有2'-脱氧假尿苷的寡脱氧核糖核苷酸之间的相互作用。将含有2'-脱氧尿苷（尿嘧啶-DNA糖基化酶的底物）的寡核苷酸在存在和不存在含有2'-脱氧假尿苷的寡核苷酸的情况下与该酶一起温育。含有2'-脱氧假尿苷的寡核苷酸存在下的反应速率明显小于寡核苷酸类似物不存在下的反应速率。结果表明，含有2'-脱氧假尿苷的寡核苷酸抑制尿嘧啶-DNA糖基化酶活性。