A re-investigation of the ribonuclease sensitivity of a DNA demethylation reaction in chicken embryo and G8 mouse myoblasts

Jp. Jost et al., A re-investigation of the ribonuclease sensitivity of a DNA demethylation reaction in chicken embryo and G8 mouse myoblasts, FEBS LETTER, 449(2-3), 1999, pp. 251-254
Citations number
Categorie Soggetti
Biochemistry & Biophysics
Journal title
ISSN journal
0014-5793 → ACNP
Year of publication
251 - 254
SICI code
Recently published results (Nucleic Acids Res, 26, 5573-5580, 1998) suggest that the ribonuclease sensitivity of the DNA demethylation reaction may be an experimental artifact due to the possible tight binding of the nuclease s to the methylated DNA substrate. Using an improved protocol we show for t wo different systems that demethylation of hemimethylated DNA is indeed sen sitive to micrococcal nuclease, requires RNA and is not an experimental art ifact. The purified 5-MeC-DNA glycosylase from chicken embryos and G8 mouse myoblasts was first incubated for 5 min at 37 degrees C with micrococcal n uclease in the presence of Ca2+ in the absence of the DNA substrate. Upon b locking the nuclease activity by the addition of 25 mM EGTA, the DNA demeth ylation reaction was initiated by adding the labeled hemimethylated DNA sub strate to the reaction mixture. Under these conditions the DNA demethylatio n reaction was abolished. In parallel controls, where the purified 5-MeC-DN A glycosylase was pre-incubated at 37 degrees C with the nuclease, Ca2+ and EGTA or with the nuclease and EGTA, RNA was not degraded and no inhibition of the demethylation reaction was obtained, As has already been shown for chicken embryos, the loss of 5-MeC-DNA glycosylase activity from G8 myoblas ts following nuclease treatment can also be restored by the addition of syn thetic RNA complementary to the methylated strand of the substrate DNA. No reactivation of 5-MeC-DNA glycosylase is obtained by complementation with a random RNA sequence, the RNA sequence complementary to the non-methylated strand or DNA, thus ruling out a non-specific competition of the RNA for th e binding of the nuclease to the labeled DNA substrate. (C) 1999 Federation of European Biochemical Societies.