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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 (11 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 non-methylated 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.

We have previously purified and characterized a 5-methylcytosine (5-MeC)-DNA glycosylase from 12 day old chick embryos [Jost,J.P. et al. (1995) J. Biol. Chem. 270, 9734-9739]. The activity of the purified enzyme is abolished upon treatment with proteinase K and ribonuclease A. RNA copurifies with 5-MeC-DNA glycosylase activity throughout all chromatographic steps and preparative gel electrophoresis. RNA with a length of approximately 300-500 nucleotides was isolated from the gel purified enzyme. Upon extensive treatment with proteinase K, the gel eluted and labeled RNA did not show any significant change in molecular mass. The purified RNA incubated alone or in the presence of Mg2+and deoxyribonucleotide phosphates had no 5-MeC-DNA glycosylase or demethylating activities. However, activity of 5-MeC-DNA glycosylase could be restored when the purified RNA was incubated with the inactive protein, free of RNA.