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

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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.