MECHANISMS OF DNA DEMETHYLATION IN CHICKEN EMBRYOS - PURIFICATION AND PROPERTIES OF A 5-METHYLCYTOSINE-DNA GLYCOSYLASE

We have previously shown that in developing chicken embryos and differentiating mouse myoblasts, the demethylation of 5-metCpGs occurs through the replacement of 5-methylcytosine by cytosine (Jest, J, P, (1993) Proc, Natl. Acad. Sci, U.S.A. 90, 4685-4688; Jest, J. P, and Jest, Y. C, (1994) J. Biol, Chem, 269, 10040-10043), We have now purified over 30,000-fold a 5-methylcytosine-DNA glycosylase from Ig-day-old chicken embryos, The enzyme copurifies with a mismatch specific thymine-DNA glycosylase and an apyrimidic-endonuclease. The reaction product of the highly purified 8-methylcytosine-DNA glycosylase is 5-methylcytosine. The copurified apyrimidic-endonuclease activity cleaves 3' from the apyrimidic sugar. A 52.5-kDa peptide, isolated as a single band from preparative SDS-polyacrylamide gels, has both the 5-methylcytosine-DNA glycosylase and the mismatch-specific thymine-DNA glycosylase activities, 5 Methylcytosine-DNA glycosylase has an apparent pi of 5.5-7.5 and maximal activity between pH 6.5 and 7.5, The K-m for hemimethylated oligonucleotide substrate is 8 x 10(-8) M with a V-max of 4 x 10(-11) mol/h/mu g protein, B-Methylcytosine-DNA glycosylase binds equally well to methylated and non-methylated DNA, The enzyme reacts six times faster with the hemimethylated DNA than with the same bifilarly methylated DNA sequence, and single-stranded methylated DNA is not a substrate, The action of the enzyme is distributive.