The Dnmt1 DNA-(cytosine-C5)-methyltransferase methylates DNA processively with high preference for hemimethylated target sites
The Dnmt1 DNA-(cytosine-C5)-methyltransferase methylates DNA processively with high preference for hemimethylated target sites
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DOI：
10.1074/jbc.m403427200
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发表时间：
2004-11-12
影响因子：
4.8
通讯作者：
Jeltsch, A
中科院分区：
文献类型：
--
作者：
Hermann, A;Goyal, R;Jeltsch, A
在细胞中，Dnmt1是DNA复制后维持DNA甲基化模式的主要酶。有证据表明，该蛋白质位于复制分叉，在那里它可以在复制后立即直接修改新生的DNA。为了阐明这一过程的潜在机制，我们在本研究中使用纯化的Dnmt1和半甲基化的底物DNA来研究DNA甲基化的过程和复制现有甲基化模式的准确性。我们证明了DNMT1甲基化一个半甲基化的958聚体底物在一个高度进行性的反应中。完全甲基化和未甲基化的CG位点不能抑制DNA的进行性甲基化。扩展之前的工作，我们表明嵌入在半甲基化背景下的未甲基化位点的修饰速度大约减少了24倍，这表明该酶准确地复制了现有的甲基化模式。完全未修饰的DNA的甲基化速度甚至更慢，这是由于含有甲基胞嘧啶的DNA对Dnmt1的变构激活。有趣的是，Dnmt1不能以过程的方式甲基化DNA不同链上的CG半甲基化位点，这表明Dnmt1保持其相对于DNA的方向，而甲基化DNA链上的CG位点。
In the cell, Dnmt1 is the major enzyme in maintenance of the pattern of DNA methylation after DNA replication. Evidence suggests that the protein is located at the replication fork, where it could directly modify nascent DNA immediately after replication. To elucidate the potential mechanism of this process, we investigate the processivity of DNA methylation and accuracy of copying an existing pattern of methylation in this study using purified Dnmt1 and hemimethylated substrate DNA. We demonstrate that Dnmt1 methylates a hemimethylated 958-mer substrate in a highly processive reaction. Fully methylated and unmethylated CG sites do not inhibit processive methylation of the DNA. Extending previous work, we show that unmethylated sites embedded in a hemimethylated context are modified at an approximately 24-fold reduced rate, which demonstrates that the enzyme accurately copies existing patterns of methylation. Completely unmodified DNA is methylated even more slowly due to an allosteric activation of Dnmt1 by methylcytosine-containing DNA. Interestingly, Dnmt1 is not able to methylate hemimethylated CG sites on different strands of the DNA in a processive manner, indicating that Dnmt1 keeps its orientation with respect to the DNA while methylating the CG sites on one strand of the DNA.