Processive methylation of hemimethylated CpG sites by mouse Dnmt1 DNA methyltransferase
Processive methylation of hemimethylated CpG sites by mouse Dnmt1 DNA methyltransferase
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DOI：
10.1074/jbc.m411126200
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发表时间：
2005-01-07
影响因子：
4.8
通讯作者：
Tajima, S
中科院分区：
文献类型：
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作者：
Vilkaitis, G;Suetake, I;Tajima, S
DNA 甲基转移酶 Dnmt1 确保复制过程中哺乳动物基因组中谱系特异性 DNA 甲基化模式的克隆传递。 Dnmt1 靶向复制灶，与 PCNA 相互作用，并有利于 CpG 位点的半甲基化形式的甲基化。为了了解其维持功能的潜在机制，我们纯化了全长 Dnmt1 的重组形式，这是一种缺乏 PCNA 和 DNA 结合位点的 Dnmt1-(291-1620) 的截短形式，并使用一系列长的未甲基化和半甲基化 DNA 底物检查了其持续合成能力。使用亚硫酸氢盐测序和发夹 PCR 技术对甲基化模式进行直接分析表明，全长 Dnmt1 以高持续性和超过 95% 的保真度对半甲基化 DNA 进行甲基化，但未甲基化 DNA 的持续性要低得多。截短形式的 Dnmt1 显示出与全长 Dnmt1 相同的特性，表明 Dnmt1 的 N 端 290 个氨基酸残基区域对于半甲基化位点的优先活性或酶的持续合成能力不是必需的。值得注意的是，我们的分析还表明，在单次持续运行期间，Dnmt1 甲基化双链 DNA 一条链上的半甲基化 CpG 位点。我们的研究结果表明，Dnmt1 的这些固有酶特性在哺乳动物基因组甲基化模式的忠实和有效维持中发挥着重要作用。
DNA methyltransferase Dnmt1 ensures clonal transmission of lineage-specific DNA methylation patterns in a mammalian genome during replication. Dnmt1 is targeted to replication foci, interacts with PCNA, and favors methylating the hemimethylated form of CpG sites. To understand the underlying mechanism of its maintenance function, we purified recombinant forms of full-length Dnmt1, a truncated form of Dnmt1-(291-1620)lacking the binding sites for PCNA and DNA and examined their processivity using a series of long unmethylated and hemimethylated DNA substrates. Direct analysis of methylation patterns using bisulfite-sequencing and hairpin-PCR techniques demonstrated that full-length Dnmt1 methylates hemimethylated DNA with high processivity and a fidelity of over 95%, but unmethylated DNA with much less processivity. The truncated form of Dnmt1 showed identical properties to full-length Dnmt1 indicating that the N-terminal 290-amino acid residue region of Dnmt1 is not required for preferential activity toward hemimethylated sites or for processivity of the enzyme. Remarkably, our analyses also revealed that Dnmt1 methylates hemimethylated CpG sites on one strand of double-stranded DNA during a single processive run. Our findings suggest that these inherent enzymatic properties of Dnmt1 play an essential role in the faithful and efficient maintenance of methylation patterns in the mammalian genome.