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

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 nucleases to the methylated DNA substrate. Using an improved protocol we show for two different systems that demethylation of hemimethylated DNA is indeed sensitive to micrococcal nuclease, requires RNA and is not an experimental artifact. The purified 5‐MeC‐DNA glycosylase from chicken embryos and G8 mouse myoblasts was first incubated for 5 min at 37°C with micrococcal nuclease in the presence of Ca2+ in the absence of the DNA substrate. Upon blocking the nuclease activity by the addition of 25 mM EGTA, the DNA demethylation reaction was initiated by adding the labeled hemimethylated DNA substrate to the reaction mixture. Under these conditions the DNA demethylation reaction was abolished. In parallel controls, where the purified 5‐MeC‐DNA glycosylase was pre‐incubated at 37°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 myoblasts following nuclease treatment can also be restored by the addition of synthetic 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 the binding of the nuclease to the labeled DNA substrate.