Nucleic Acids Research, Vol 25, Issue 22 4545-4550, Copyright © 1997 by Oxford University Press
JP Jost, M Fremont, M Siegmann and J Hofsteenge
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.
ARTICLES
The RNA moiety of chick embryo 5-methylcytosine- DNA glycosylase targets DNA demethylation
Friedrich Miescher Institute, PO Box 2543, CH-4002 Basel, Switzerland. jost@fmi.ch
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