MECHANISMS OF DNA DEMETHYLATION IN CHICKEN EMBRYOS - PURIFICATION AND PROPERTIES OF A 5-METHYLCYTOSINE-DNA GLYCOSYLASE
MECHANISMS OF DNA DEMETHYLATION IN CHICKEN EMBRYOS - PURIFICATION AND PROPERTIES OF A 5-METHYLCYTOSINE-DNA GLYCOSYLASE
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DOI：
10.1074/jbc.270.17.9734
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发表时间：
1995-04-28
影响因子：
4.8
通讯作者：
LEUNG, R
中科院分区：
文献类型：
--
作者：
JOST, JP;SIEGMANN, M;LEUNG, R
我们先前已经表明，在发育鸡胚胎和分化小鼠成肌细胞中，5-metCpG的去甲基化通过胞嘧啶取代5-甲基胞嘧啶而发生（Jest，J，P，（1993）Proc，Natl. Acad. Sci，U. S. A. 90，4685-4688; Jest，J. P和Jest，Y. C，（1994）J. Biol，Chem，269，10040-10043），我们现在已经从Ig日龄的鸡胚中纯化了超过30，000倍的5-甲基胞嘧啶-DNA糖基化酶。该酶与错配特异性胸腺嘧啶-DNA糖基化酶和无嘧啶核酸内切酶共纯化。高度纯化的8-甲基胞嘧啶-DNA糖基化酶的反应产物是5-甲基胞嘧啶。共纯化的无嘧啶核酸内切酶活性从无嘧啶糖切割3'。从制备的SDS-聚丙烯酰胺凝胶中分离的单一条带的52.5-kDa肽具有5-甲基胞嘧啶-DNA糖基化酶和错配特异性胸腺嘧啶-DNA糖基化酶活性，5-甲基胞嘧啶-DNA糖基化酶具有5.5-7.5的表观pi和在pH6.5 - 7.5之间的最大活性，半甲基化寡核苷酸底物的Km为8 × 10（-8）M，V-max为4 × 10（-11）mol/h/μ g蛋白质，B-甲基胞嘧啶-DNA糖基化酶与甲基化和非甲基化DNA的结合同样良好，该酶与半甲基化DNA的反应速度比与相同的双股甲基化DNA序列的反应速度快6倍，并且单链甲基化DNA不是底物。
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.