Enzymatic analysis of Tet proteins: key enzymes in the metabolism of DNA methylation.
Enzymatic analysis of Tet proteins: key enzymes in the metabolism of DNA methylation.
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DOI：
10.1016/b978-0-12-391940-3.00005-6
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发表时间：
2012
影响因子：
--
通讯作者：
Zhang, Yi
中科院分区：
文献类型：
--
作者：
Shen, Li;Zhang, Yi
表观遗传学领域最令人兴奋的最新进展之一是发现DNA中的5-甲基胞嘧啶（5 mC）可以被称为泰特蛋白的蛋白质家族反复氧化，生成5-羟甲基胞嘧啶（5 hmC）、5-甲酰基胞嘧啶（5 fC）和5-羧基胞嘧啶（5caC）。这些5 mC衍生物可以通过胸腺嘧啶-DNA糖基化酶（TDG）进一步加工，然后通过碱基切除修复或通过复制依赖性稀释导致DNA去甲基化。由于5 mC与其氧化衍生物之间的相似性，许多用于5 mC分析的常规技术无法区分5 mC和5 hmC/5 fC/5caC。在这里，我们描述了2D-TLC和质谱法，我们已经成功地用于区分5 mC从其氧化衍生物，以及在表征的酶活性的泰特蛋白在体外和体内。
One of the most exciting recent advances in the epigenetic field is the discovery that 5-methylcytosine (5mC) in DNA can be iteratively oxidized by a family of proteins known as Tet proteins to generate 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC). These 5mC derivatives can be further processed by thymine-DNA glycosylase (TDG) followed by base excision repair or by replication-dependent dilution leading to DNA demethylation. Given the similarity between 5mC and its oxidation derivatives, many of the conventional techniques used for 5mC analysis cannot distinguish between 5mC and 5hmC/5fC/5caC. Here, we describe 2D-TLC and mass spectrometry methods that we have successfully used in differentiating 5mC from its oxidative derivatives as well as in characterizing the enzymatic activity of Tet proteins both in vitro and in vivo.