Nucleic Acids Research, Vol 26, Issue 24 5573-5580, Copyright © 1998 by Oxford University Press
JF Swisher, E Rand, H Cedar and A Marie Pyle
The mechanism for demethylation of DNA in rat myoblasts has recently been
studied using a new in vitro system that monitors demethylation in whole
cell extracts. Previous investigations using this system had indicated that
demethylation is resistant to conditions that are normally assumed to
denature or digest proteins. Remarkably, it was reported that the activity
appeared to be sensitive to the action of ribonuclease, suggesting a role
for RNA in the demethylation of DNA. This manuscript reports that, upon
further purification of the extract, demethylation activity has properties
that are different. When subjected to more rigorous procedures for
digestion of proteins, the demethylase activity disappears. Furthermore,
RNase sensitivity of the extract disappears when a quantity of unmethylated
competitor DNA is added to the reaction mix or when extracts treated with
RNase are subsequently treated with protease. Although a role for RNA
cannot be completely discounted, it is unlikely that this demethyl-ation
reaction involves RNA cofactors or ribozyme components. These results have
important implications for the mechanism of DNA demethylation and they
exemplify the potential pitfalls of experiments in which new biological
roles for RNA are evaluated using RNase sensitivity experiments.
ARTICLES
Analysis of putative RNase sensitivity and protease insensitivity of demethylation activity in extracts from rat myoblasts
Department of Biochemistry and Molecular Biophysics, Columbia University and Howard Hughes Medical Institute, New York, NY 10032, USA. harri.hakala@utu.fi
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