ACTIVATION OF MAMMALIAN DNA METHYLTRANSFERASE BY CLEAVAGE OF A ZN BINDING REGULATORY DOMAIN
ACTIVATION OF MAMMALIAN DNA METHYLTRANSFERASE BY CLEAVAGE OF A ZN BINDING REGULATORY DOMAIN
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DOI：
10.1002/j.1460-2075.1992.tb05326.x
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发表时间：
1992-07-01
期刊：
影响因子：
11.4
通讯作者：
BESTOR, TH
中科院分区：
文献类型：
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作者：
BESTOR, TH
哺乳动物DNA（胞嘧啶-5）甲基转移酶含有一个与细菌胞嘧啶-5限制性甲基转移酶密切相关的c端结构域。这个甲基转移酶结构域连接到一个大的n端结构域。结果表明，N端结构域含有一个Zn结合位点，N端结构域和c端结构域可以通过胰蛋白酶或金黄色葡萄球菌蛋白酶V8裂解分离；通过Edman降解确定蛋白酶V8的裂解位点位于连接两个结构域的赖氨酸和甘酰基交替序列的10个残基c端，以及哺乳动物和细菌胞嘧啶甲基转移酶之间保守的第一序列基序的6个残基n端。虽然完整的酶在未甲基化的DNA底物上几乎没有活性，但结构域之间的切割会大大刺激未甲基化DNA的初始甲基化速度，而对半甲基化DNA的甲基化速度没有实质性的改变。这些发现表明，DNA甲基转移酶的n端结构域通过抑制c端结构域的新生活性来确保甲基化模式的克隆繁殖。哺乳动物DNA甲基转移酶很可能是由一种类似原核生物的限制性甲基转移酶和一种不相关的DNA结合蛋白融合而产生的。体内蛋白水解裂解对DNA甲基转移酶新生活性的刺激可能有助于在衰老动物、肿瘤和培养细胞系中观察到的DNA异位甲基化过程。
Mammalian DNA (cytosine-5) methyltransferase contains a C-terminal domain that is closely related to bacterial cytosine-5 restriction methyltransferases. This methyltransferase domain is linked to a large N-terminal domain. It is shown here that the N-terminal domain contains a Zn binding site and that the N- and C-terminal domains can be separated by cleavage with trypsin or Staphylococcus aureus protease V8; the protease V8 cleavage site was determined by Edman degradation to lie 10 residues C-terminal of the run of alternating lysyl and glycyl residues which joins the two domains and six residues N-terminal of the first sequence motif conserved between the mammalian and bacterial cytosine methyltransferases. While the intact enzyme had little activity on unmethylated DNA substrates, cleavage between the domains caused a large stimulation of the initial velocity of methylation of unmethylated DNA without substantial change in the rate of methylation of hemimethylated DNA. These findings indicate that the N-terminal domain of DNA methyltransferase ensures the clonal propagation of methylation patterns through inhibition of the de novo activity of the C-terminal domain. Mammalian DNA methyltransferase is likely to have arisen via fusion of a prokaryotic-like restriction methyltransferase and an unrelated DNA binding protein. Stimulation of the de novo activity of DNA methyltransferase by proteolytic cleavage in vivo may contribute to the process of ectopic methylation observed in the DNA of aging animals, tumors and in lines of cultured cells.