NUCLEAR EXTRACTS OF CHICKEN EMBRYOS PROMOTE AN ACTIVE DEMETHYLATION OF DNA BY EXCISION REPAIR OF 5-METHYLDEOXYCYTIDINE

Here I show that nuclear extracts of chicken embryos can promote the a ctive demethylation of DNA. The evidence shows that in hemimethylated DNA (i.e., methylated on one strand only) demethylation of 5mCpG occur s through nucleotide excision repair. The first step of demethylation is the formation of specific nicks 5' from 5-methyldeoxycytidine. Nick s are also observed in vitro on symmetrically methylated CpGs (i.e., m ethylated on both strands) but they result in breakage of the oligonuc leotide with no repair. No specific nicks are observed on the nonmethy lated CpG. Nicks are strictly 5mCpG specific and do not occur on 5mCpC , 5mCpT, 5mCpA, or 6mApT. The effect of nonspecific nuclease(s) has be en 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 ac tion by DNA polymerase beta. In extracts of chicken embryos, the excis ion repair of mCpG is highest between the 6th and the 12th day of deve lopment, whereas it is barely detectable in nuclear extracts from diff erent organs of adults. The possible implications of 5mCpG endonucleas e activity in active demethylation of DNA during differentiation is di scussed.