THE RNA MOIETY OF CHICK-EMBRYO 5-METHYLCYTOSINE-DNA GLYCOSYLASE TARGETS DNA DEMETHYLATION

We have previously shown that DNA demethylation by chick embryo 5-meth ylcytosine (5-MeC)-DNA glycosylase needs both protein and RNA. RNA fro m 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 n ot have any sequence homology with any other known RNA. RNase-inactiva ted 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 sequen ces complementary to the labeled DNA substrate are present in the reco mbinant RNA. Small synthetic oligoribonucleotides (II bases long) comp lementary to the region of methylated CpGs of the hemimethylated doubl e-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 t he same DNA-substrate are inactive when incubated in the complementati on 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 r estore 5-MeC-DNA glycosylase activity.-An excess of targeting oligorib onucleotides cannot change the preferential substrate specificity of t he enzyme for hemimethylated double-stranded DNA.