在5-甲基胞嘧啶的氧化形式存在下维持DNA甲基转移酶活性：十一个十一易位介导的DNA脱甲基化的结构基础,Biochemistry

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在5-甲基胞嘧啶的氧化形式存在下维持DNA甲基转移酶活性：十一个十一易位介导的DNA脱甲基化的结构基础
Biochemistry ( IF 2.9 ) Pub Date : 2018-09-19 00:00:00 , DOI: 10.1021/acs.biochem.8b00683
Christopher L. Seiler ₁ , Jenna Fernandez ₁ , Zoe Koerperich ₁ , Molly P. Andersen ₁ , Delshanee Kotandeniya ₁ , Megin E. Nguyen ₂ , Yuk Y. Sham _{2,

3} , Natalia Y. Tretyakova ₁

Affiliation

DNA甲基化和去甲基化的精确平衡是细胞身份，发育和生长的表观遗传控制所必需的。从头开始引入DNA甲基化标记DNA甲基转移酶DNMT3a / b，并在整个细胞分裂过程中被DNA甲基转移酶1（DNMT1）维持，DNA甲基转移酶1向DNA复制过程中产生的半甲基化CpG二核苷酸添加了甲基。十一个11易位（TET）双加氧酶将5-甲基胞嘧啶（mC）氧化为5-羟甲基胞嘧啶（hmC），5-甲酰基胞嘧啶（fC）和5-羧基胞嘧啶（caC），该过程已知可诱导DNA脱甲基化和基因重新激活。在这项研究中，我们研究了在氧化形式的mC存在下人DNMT1的催化活性。基于质谱的分析方法用于研究DNMT1介导的CG二核苷酸在目标胞嘧啶中含有C，mC，hmC，fC或caC的胞嘧啶甲基化动力学。同源性建模，再加上分子动力学模拟，用于研究mC氧化对蛋白质-DNA复合物的几何结构的影响。DNMT1的酶活性受mC氧化状态的强烈影响，催化效率按以下顺序降低：mC> hmC> fC> caC。分子动力学模拟显示，DNMT1与含有氧化形式的mC的DNA双链体形成了一种无效的复合物，这是由于该蛋白质的靶标识别域与胞嘧啶上C-5取代基相互作用的结果。我们的结果为TET介导的DNA脱甲基化提供了新的结构和机理见解。mC> hmC> fC> caC。分子动力学模拟显示，DNMT1与含有氧化形式的mC的DNA双链体形成了一种无效的复合物，这是由于该蛋白质的靶标识别域与胞嘧啶上C-5取代基相互作用的结果。我们的结果为TET介导的DNA脱甲基化提供了新的结构和机理见解。mC> hmC> fC> caC。分子动力学模拟显示，DNMT1与含有氧化形式的mC的DNA双链体形成了一种无效的复合物，这是由于该蛋白质的靶标识别域与胞嘧啶上C-5取代基相互作用的结果。我们的结果为TET介导的DNA脱甲基化提供了新的结构和机理见解。

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Maintenance DNA Methyltransferase Activity in the Presence of Oxidized Forms of 5-Methylcytosine: Structural Basis for Ten Eleven Translocation-Mediated DNA Demethylation

A precise balance of DNA methylation and demethylation is required for epigenetic control of cell identity, development, and growth. DNA methylation marks are introduced by de novo DNA methyltransferases DNMT3a/b and are maintained throughout cell divisions by DNA methyltransferase 1 (DNMT1), which adds methyl groups to hemimethylated CpG dinucleotides generated during DNA replication. Ten eleven translocation (TET) dioxygenases oxidize 5-methylcytosine (mC) to 5-hydroxymethylcytosine (hmC), 5-formylcytosine (fC), and 5-carboxylcytosine (caC), a process known to induce DNA demethylation and gene reactivation. In this study, we investigated the catalytic activity of human DNMT1 in the presence of oxidized forms of mC. A mass spectrometry-based assay was employed to study the kinetics of DNMT1-mediated cytosine methylation in CG dinucleotides containing C, mC, hmC, fC, or caC across from the target cytosine. Homology modeling, coupled with molecular dynamics simulations, was used to explore the structural consequences of mC oxidation with regard to the geometry of protein–DNA complexes. The DNMT1 enzymatic activity was strongly affected by the oxidation status of mC, with the catalytic efficiency decreasing in the following order: mC > hmC > fC > caC. Molecular dynamics simulations revealed that DNMT1 forms an unproductive complex with DNA duplexes containing oxidized forms of mC as a consequence of altered interactions of the target recognition domain of the protein with the C-5 substituent on cytosine. Our results provide new structural and mechanistic insight into TET-mediated DNA demethylation.

更新日期：2018-09-19

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