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 (Jost, J. P. (1993) Proc. Natl. Acad. Sci. U.S.A. 90, 4685-4688; Jost, J. P. & Jost, Y.C. (1994) J. Biol. Chem. 269, 10040-10043). We have now purified over 30,000-fold a 5-methylcytosine-DNA glycosylase from 12-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 5-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 Km for hemimethylated oligonucleotide substrate is 8 x 10(-8) M with a Vmax of 4 x 10(-11) mol/h/micrograms proteins. 5-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.
Jost
J P
JP
Friedrich Miescher Institute, Basel, Switzerland.
Siegmann
M
M
Sun
L
L
Leung
R
R
D000818:Animals; D001483:Base Sequence; D002642:Chick Embryo; D004247:DNA / Q000378:metabolism*; D045647:DNA Glycosylases; D008745:Methylation; D008969:Molecular Sequence Data; D009699:N-Glycosyl Hydrolases / Q000302:isolation & purification / Q000378:metabolism*
D004247:DNA; C093335:5-methylcytosine-DNA glycosylase; D045647:DNA Glycosylases; D009699:N-Glycosyl Hydrolases