Morales-Ruiz Teresa, Ortega-Galisteo Ana Pilar, Ponferrada-Marín María Isabel, Martínez-Macías María Isabel, Ariza Rafael R, Roldán-Arjona Teresa
Departamento de Genética, Universidad de Córdoba, 14071 Córdoba, Spain.
Proc Natl Acad Sci U S A. 2006 May 2;103(18):6853-8. doi: 10.1073/pnas.0601109103. Epub 2006 Apr 19.
Cytosine methylation is an epigenetic mark that promotes gene silencing and plays important roles in development and genome defense against transposons. Methylation patterns are established and maintained by DNA methyltransferases that catalyze transfer of a methyl group from S-adenosyl-L-methionine to cytosine bases in DNA. Erasure of cytosine methylation occurs during development, but the enzymatic basis of active demethylation remains controversial. In Arabidopsis thaliana, DEMETER (DME) activates the maternal expression of two imprinted genes silenced by methylation, and REPRESSOR OF SILENCING 1 (ROS1) is required for release of transcriptional silencing of a hypermethylated transgene. DME and ROS1 encode two closely related DNA glycosylase domain proteins, but it is unknown whether they participate directly in a DNA demethylation process or counteract silencing through an indirect effect on chromatin structure. Here we show that DME and ROS1 catalyze the release of 5-methylcytosine (5-meC) from DNA by a glycosylase/lyase mechanism. Both enzymes also remove thymine, but not uracil, mismatched to guanine. DME and ROS1 show a preference for 5-meC over thymine in the symmetric dinucleotide CpG context, where most plant DNA methylation occurs. Nevertheless, they also have significant activity on both substrates at CpApG and asymmetric sequences, which are additional methylation targets in plant genomes. These findings suggest that a function of ROS1 and DME is to initiate erasure of 5-meC through a base excision repair process and provide strong biochemical evidence for the existence of an active DNA demethylation pathway in plants.
胞嘧啶甲基化是一种表观遗传标记，可促进基因沉默，并在发育以及基因组对转座子的防御中发挥重要作用。甲基化模式由DNA甲基转移酶建立和维持，这些酶催化甲基从S-腺苷-L-甲硫氨酸转移至DNA中的胞嘧啶碱基上。胞嘧啶甲基化的消除发生在发育过程中，但主动去甲基化的酶学基础仍存在争议。在拟南芥中，DNA糖基化酶DEMETER（DME）激活两个因甲基化而沉默的印记基因的母本表达，而超甲基化转基因转录沉默的释放则需要沉默抑制因子1（ROS1）。DME和ROS1编码两种密切相关的具有DNA糖基化酶结构域的蛋白质，但尚不清楚它们是直接参与DNA去甲基化过程，还是通过对染色质结构的间接作用来对抗基因沉默。在此我们表明，DME和ROS1通过糖基化酶/裂合酶机制催化从DNA中释放5-甲基胞嘧啶（5-meC）。这两种酶还能去除与鸟嘌呤错配的胸腺嘧啶，但不能去除尿嘧啶。在大多数植物DNA甲基化发生的对称二核苷酸CpG环境中，DME和ROS1对5-meC的偏好高于胸腺嘧啶。然而，它们在CpApG和不对称序列这两种植物基因组中的额外甲基化靶点上，对两种底物也都具有显著活性。这些发现表明，ROS1和DME的功能是通过碱基切除修复过程启动5-meC的消除，并为植物中存在主动DNA去甲基化途径提供了有力的生化证据。
