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J Biol Chem. 1995 Apr 28;270(17):9734-9.

Mechanisms of DNA demethylation in chicken embryos. Purification and properties of a 5-methylcytosine-DNA glycosylase.

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  • 1Friedrich Miescher Institute, Basel, Switzerland.

Abstract

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.

PMID:
7730351
[PubMed - indexed for MEDLINE]
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