CXXC domain of human DNMT1 is essential for enzymatic activity
CXXC domain of human DNMT1 is essential for enzymatic activity
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DOI：
10.1021/bi8011725
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发表时间：
2008-09-23
期刊：
影响因子：
2.9
通讯作者：
Pradhan, Sriharsa
中科院分区：
文献类型：
--
作者：
Pradhan, Mihika;Esteve, Pierre-Olivier;Pradhan, Sriharsa
DNA胞嘧啶甲基化是人类基因组中由DNA甲基转移酶促进的主要表观遗传基因沉默标记之一。 DNA 胞嘧啶 5 甲基转移酶 1 (DNMT1) 在体细胞中执行维持甲基化。在癌细胞中，DNMT1 负责 CpG 岛的异常高甲基化和抑癌基因的沉默。在这里，我们表明，具有催化活性的重组DNMT1（从氨基末端缺少580个氨基酸）与非甲基化DNA的结合比半甲基化或甲基化DNA具有更高的亲和力。为了进一步了解酶的结合域，我们使用了凝胶位移测定。我们已经证明 DNMT1 的 CXXC 区域（C 是半胱氨酸；X 是任何氨基酸）与未甲基化的 CpG 二核苷酸特异性结合。此外，保守半胱氨酸的突变消除了 CXXC 介导的 DNA 结合。在转染的 COS-7 细胞中，CXXC 删除了位于复制灶上的 DNMT1 (DNMTI1(Delta CXXC))。点突变体和 DNMT1(Delta CXXC) 酶均表现出催化活性显着降低，证实该结构域对于酶活性至关重要。尽管存在内源性野生型酶，但具有 DNMT1(Delta CXXC) 的永久细胞系在 rDNA 位点上显示出部分基因组甲基化缺失。因此，包含 DNMT1 氨基末端区域的 CXXC 结构域与 DNA 甲基转移酶活性的催化结构域配合。
DNA cytosine methylation is one of the major epigenetic gene silencing marks in the human genome facilitated by DNA methyltransferases. DNA cytosine-5 methyltransferase 1 (DNMT1) performs maintenance methylation in somatic cells. In cancer cells, DNMT1 is responsible for the aberrant hypermethylation of CpG islands and the silencing of tumor suppressor genes. Here we show that the catalytically active recombinant DNMT1, lacking 580 amino acids from the amino terminus, binds to unmethylated DNA with higher affinity than hemimethylated or methylated DNA. To further understand the binding domain of enzyme, we have used gel shift assay. We have demonstrated that the CXXC region (C is cysteine; X is any amino acid) of DNMT1 bound specifically to unmethylated CpG dinucleotides. Furthermore, mutation of the conserved cysteines abolished CXXC mediated DNA binding. In transfected COS-7 cells, CXXC deleted DNMT1 (DNMTI1(Delta CXXC)) localized on replication foci. Both point mutant and DNMT1(Delta CXXC) enzyme displayed significant reduction in catalytic activity, confirming that this domain is crucial for enzymatic activity. A permanent cell line with DNMT1(Delta CXXC) displayed partial loss of genomic methylation on rDNA loci, despite the presence of endogenous wild-type enzyme. Thus, the CXXC domain encompassing the amino terminus region of DNMT1 cooperates with the catalytic domain for DNA methyltransferase activity.