The RNA moiety of chick embryo 5-methylcytosine-DNA glycosylase targets DNA demethylation
The RNA moiety of chick embryo 5-methylcytosine-DNA glycosylase targets DNA demethylation
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DOI：
10.1093/nar/25.22.4545
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发表时间：
1997-11-15
影响因子：
14.9
通讯作者：
Hofsteenge, J
中科院分区：
文献类型：
--
作者：
Jost, JP;Fremont, M;Hofsteenge, J
我们以前已经表明，DNA去甲基化的鸡胚5-甲基胞嘧啶（5-MeC）-DNA糖基化酶需要蛋白质和RNA。从通过SDS-PAGE纯化的酶中分离并克隆RNA。克隆具有范围从240至670 bp的插入物，并且平均每14个碱基含有一个CpG;测试的所有六个克隆具有不同的序列，并且与任何其他已知RNA没有任何序列同源性。RNA酶失活的5-MeC-DNA糖基化酶在与重组RNA孵育时恢复酶活性。然而，当重组RNA与DNA底物单独孵育时，没有去甲基化活性。与标记的DNA底物互补的短序列存在于重组RNA中。与半甲基化双链DNA底物的甲基化CpG区域互补的小的合成寡核糖核苷酸（II碱基长）恢复RNA酶失活的5-MeC-DNA糖基化酶的活性。在互补试验中孵育时，与相同DNA底物的非甲基化链互补的相应寡脱氧核糖核苷酸或寡核糖核苷酸无活性。与CpG靶序列互补的最少4个碱基对于RNA酶处理的5-MeC-DNA糖基化酶的再活化是必需的。与双链寡核糖核苷酸互补不能恢复5-MeC-DNA糖基化酶活性。过量的靶向寡核糖核苷酸不能改变酶对半甲基化双链DNA的优先底物特异性。
We have previously shown that DNA demethylation by chick embryo 5-methylcytosine (5-MeC)-DNA glycosylase needs both protein and RNA. RNA from enzyme purified by SDS-PAGE was isolated and cloned. The clones have an insert ranging from 240 to 670 bp and contained on average one CpG per 14 bases; All six clones tested had different sequences and did not have any sequence homology with any other known RNA. RNase-inactivated 5-MeC-DNA glycosylase regained enzyme activity when incubated with recombinant RNA. However, when recombinant RNA was incubated with the DNA substrate alone there was no demethylation activity. Short sequences complementary to the labeled DNA substrate are present in the recombinant RNA. Small synthetic oligoribonucleotides (II bases long) complementary to the region of methylated CpGs of the hemimethylated double-stranded DNA substrate restore the activity of the RNase-inactivated 5-MeC-DNA glycosylase. The corresponding oligodeoxyribonucleotide or the oligoribonucleotide complementary to the nonmethylated strand of the same DNA-substrate are inactive when incubated in the complementation test. A minimum of 4 bases complementary to the CpG target sequence are necessary for reactivation of RNase-treated 5-MeC-DNA glycosylase. Complementation with double-stranded oligoribonucleotides does not restore 5-MeC-DNA glycosylase activity.-An excess of targeting oligoribonucleotides cannot change the preferential substrate specificity of the enzyme for hemimethylated double-stranded DNA.