细胞和组织中的O~6-甲基脱氧鸟嘌呤核苷的脱甲基作用

为了探讨细胞和组织中的O~6-甲基脱氧鸟嘌呤核苷的脱甲基作用,我们合成了~3H]O~6-mGua DNA、O~6-mdGuo、O~6-mdGMP、O~6-mdGTP等底物,在体外利用高压液相色谱分析细胞和组织提取物去除鸟嘌呤基团第六位氧原子上甲基的能力。结果表明,在细胞和组织提取物中存在一种去甲基酶,它能去除O~6-甲基脱氧鸟嘌呤核苷、O~6-甲基脱氧鸟嘌呤核苷-磷酸上的甲基,生成脱氧鸟嘌呤核苷或脱氧鸟嘌呤核苷-磷酸,并伴随着甲醇的释放。它不同于DNA甲基转移酶。没有观察到它对O~6-甲基鸟嘌呤DNA、O~6-甲基脱氧鸟嘌呤核苷三磷酸、O~4-甲基胸腺嘧啶核苷以及O~6-甲基鸟嘌呤的去甲基作用。

O~6-mdGuo DEMETHYLATION IN CELLS AND TISSUES

We synthesized some substrates,such as ~3H] O~6-mGua DNA,O~6-mdGuo,O~6-mdGMP,O~6-mdGTP etc.and used the method of HPLC to study the demethylation of cells and tissus in vitro. The results indicated that there is a de-methylase in the extract of cells and tissuse.It can remove the methyl group from O~6-mdGuo and O~6-mdGMP,to yield dGuo and dGMP respectively.The disappearance of methyl group from O~6-mdGuo and O~6-mdGMP is accompanied by the formation of CH_3OH.It was not observed that demethylase rcacted with O~6-mGua DNA,O~6-mGua and O~4-mdT.The demethylase is different from O~6-methylguanine DNA methyltransferase in the substrates reacted with enzymes and the mechanism of removing methyl group.The data show that the demethylase exists in Hela SMR cells without any O~6-methylguanine DNA methyltransferase activity.When cells were treated by MNNG,no obvious change on the activity of demethylase in cells was observed.A hypothesis on the biological function of the demethylase is proposed.