Analysis of putative RNase sensitivity and protease insensitivity of demethylation activity in extracts from rat myoblasts

The mechanism for demethylation of DNA in rat myoblasts has recently been s tudied using a new in vitro system that monitors demethylation in whole cel l extracts. Previous investigations using this system had indicated that de methylation is resistant to conditions that are normally assumed to denatur e or digest proteins. Remarkably, it was reported that the activity appeare d to be sensitive to the action of ribonuclease, suggesting a role for RNA in the demethylation of DNA, This manuscript reports that, upon further pur ification of the extract, demethylation activity has properties that are di fferent. When subjected to more rigorous procedures for digestion of protei ns, the demethylase activity disappears. Furthermore, RNase sensitivity of the extract disappears when a quantity of unmethylated competitor DNA is ad ded to the reaction mix or when extracts treated with RNase are subsequentl y treated with protease. Although a role for RNA cannot be completety disco unted, it is unlikely that this demethylation reaction involves RNA cofacto rs or ribozyme components. These results have important implications for th e mechanism of DNA demethylation and they exemplify the potential pitfalls of experiments in which new biological roles for RNA are evaluated using RN ase sensitivity experiments.