NUCLEAR EXTRACTS OF CHICKEN EMBRYOS PROMOTE AN ACTIVE DEMETHYLATION OF DNA BY EXCISION REPAIR OF 5-METHYLDEOXYCYTIDINE
NUCLEAR EXTRACTS OF CHICKEN EMBRYOS PROMOTE AN ACTIVE DEMETHYLATION OF DNA BY EXCISION REPAIR OF 5-METHYLDEOXYCYTIDINE
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DOI：
10.1073/pnas.90.10.4684
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发表时间：
1993-05-15
影响因子：
11.1
通讯作者：
JOST, JP
中科院分区：
文献类型：
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作者：
JOST, JP
在这里，我表明，鸡胚的核提取物可以促进DNA的主动去甲基化。证据表明，在半甲基化DNA中（即，仅在一条链上甲基化）5 mCpG的去甲基化通过核苷酸切除修复发生。去甲基化的第一步是从5-甲基脱氧胞苷5'形成特异性切口。在体外也在对称甲基化的CpG（即，在两条链上甲基化），但它们导致寡核苷酸断裂而没有修复。在非甲基化CpG上未观察到特异性切口。切口是严格的5 mCpG特异性的，并且不发生在5 mCpC、5 mCpT、5 mCpA或6 mApT上。已排除非特异性核酸酶的影响。mCpG的切口在20 mM EDTA存在下发生，而与5 mCpG周围序列的性质无关。未检测到甲基胞嘧啶糖基化酶活性。该修复是阿非迪霉素和N-乙基马来酰亚胺抗性的，表明DNA聚合酶β的修复作用。在鸡胚胎提取物中，mCpG的切除修复在发育的第6天和第12天之间最高，而在来自成人不同器官的核提取物中几乎检测不到。5 mCpG核酸内切酶活性在分化过程中的DNA主动去甲基化的可能影响进行了讨论。
Here I show that nuclear extracts of chicken embryos can promote the active demethylation of DNA. The evidence shows that in hemimethylated DNA (i.e., methylated on one strand only) demethylation of 5mCpG occurs through nucleotide excision repair. The first step of demethylation is the formation of specific nicks 5' from 5-methyldeoxycytidine. Nicks are also observed in vitro on symmetrically methylated CpGs (i.e., methylated on both strands) but they result in breakage of the oligonucleotide with no repair. No specific nicks are observed on the nonmethylated CpG. Nicks are strictly 5mCpG specific and do not occur on 5mCpC, 5mCpT, 5mCpA, or 6mApT. The effect of nonspecific nuclease(s) has been ruled out. The nicking of mCpG takes place in the presence of 20 mM EDTA irrespective of the nature of the sequence surrounding the 5mCpG. No methylcytosine glycosylase activity could be detected. The repair is aphidicolin and N-ethylmaleimide resistant, suggesting a repair action by DNA polymerase beta. In extracts of chicken embryos, the excision repair of mCpG is highest between the 6th and the 12th day of development, whereas it is barely detectable in nuclear extracts from different organs of adults. The possible implications of 5mCpG endonuclease activity in active demethylation of DNA during differentiation is discussed.