Department of Chemistry , University of North Texas , Denton , Texas 76201 , United States.
Biochemistry. 2019 Feb 5;58(5):411-421. doi: 10.1021/acs.biochem.8b00912. Epub 2018 Nov 14.
Enzymes of the ten-eleven translocation (TET) family add diversity to the repertoire of nucleobase modifications by catalyzing the oxidation of 5-methylcytosine (5mC). TET enzymes were initially found to oxidize 5-methyl-2'-deoxycytidine in genomic DNA, yielding products that contribute to epigenetic regulation in mammalian cells, but have since been found to also oxidize 5-methylcytidine in RNA. Considering the different configurations of single-stranded (ss) and double-stranded (ds) DNA and RNA that coexist in a cell, defining the scope of TET's preferred activity and the mechanisms of substrate selectivity is critical to better understand the enzymes' biological functions. To this end, we have systematically examined the activity of human TET2 on DNA, RNA, and hybrid substrates in vitro. We found that, while ssDNA and ssRNA are well tolerated, TET2 is most proficient at dsDNA oxidation and discriminates strongly against dsRNA. Chimeric and hybrid substrates containing mixed DNA and RNA character helped reveal two main features by which the enzyme discriminates between substrates. First, the identity of the target nucleotide alone is the strongest reactivity determinant, with a preference for 5-methyldeoxycytidine, while both DNA or RNA are relatively tolerated on the rest of the target strand. Second, while a complementary strand is not required for activity, DNA is the preferred partner, and complementary RNA diminishes reactivity. Our biochemical analysis, complemented by molecular dynamics simulations, provides support for an active site optimally configured for dsDNA reactivity but permissive for various nucleic acid configurations, suggesting a broad range of plausible roles for TET-mediated 5mC oxidation in cells.
TET 家族的酶通过催化 5-甲基胞嘧啶（5mC）的氧化，为核碱基修饰的多样性做出贡献。TET 酶最初被发现可以氧化基因组 DNA 中的 5-甲基-2'-脱氧胞苷，生成的产物有助于哺乳动物细胞中的表观遗传调控，但后来发现它们也可以氧化 RNA 中的 5-甲基胞嘧啶。考虑到细胞中同时存在单链（ss）和双链（ds）DNA 和 RNA 的不同构象，定义 TET 优先活性的范围和底物选择性的机制对于更好地理解酶的生物学功能至关重要。为此，我们系统地研究了人 TET2 在体外对 DNA、RNA 和杂交底物的活性。我们发现，虽然 ssDNA 和 ssRNA 可以很好地耐受，但 TET2 最擅长 dsDNA 氧化，并且强烈区分 dsRNA。包含混合 DNA 和 RNA 特征的嵌合和杂交底物有助于揭示酶区分底物的两个主要特征。首先，仅靶核苷酸的身份是最强的反应性决定因素，对 5-甲基脱氧胞苷有偏好，而 DNA 或 RNA 在其余靶链上相对耐受。其次，尽管活性不需要互补链，但 DNA 是首选伴侣，互补 RNA 会降低反应性。我们的生化分析，辅以分子动力学模拟，为活性位点最适合 dsDNA 反应性但允许各种核酸构象的配置提供了支持，这表明 TET 介导的 5mC 氧化在细胞中可能具有广泛的作用。
