The DNA Repair Protein XRCC1 Functions in the Plant DNA Demethylation Pathway by Stimulating Cytosine Methylation (5-meC) Excision, Gap Tailoring, and DNA Ligation
The DNA Repair Protein XRCC1 Functions in the Plant DNA Demethylation Pathway by Stimulating Cytosine Methylation (5-meC) Excision, Gap Tailoring, and DNA Ligation
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DOI：
10.1074/jbc.m112.427617
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发表时间：
2013-02-22
影响因子：
4.8
通讯作者：
Roldan-Arjona, Teresa
中科院分区：
文献类型：
--
作者：
Isabel Martinez-Macias, Maria;Cordoba-Canero, Dolores;Roldan-Arjona, Teresa
DNA甲基化模式是拮抗剂甲基化和去甲基化机制的动态结果，但后者仍然知之甚少。植物中主动的DNA去甲基化是由一个以拟南芥ROS 1（沉默阻遏物1）为代表的DNA糖基化酶家族介导的。ROS 1及其同源物去除5-甲基胞嘧啶并在脱碱基位点切割糖骨架，从而启动碱基切除修复途径，最终插入未甲基化的胞嘧啶。DNA 3 '-磷酸酶ZDP处理由ROS 1产生的一些切割产物，允许随后的DNA聚合和连接步骤。在这项工作中，我们研究了植物XRCC 1（X射线交叉互补组蛋白1）在DNA去甲基化中的可能作用。我们发现XRCC 1在体外与ROS 1和ZDP相互作用，并刺激这两种蛋白质的酶活性。此外，从xrcc 1突变体植物的提取物表现出降低的能力，以完成由ROS 1启动的DNA去甲基化。抗XRCC 1抗体抑制去甲基化过程中产生的单核苷酸缺口中的阻断性3 ′-磷酸的去除，并降低拟南芥细胞提取物连接有切口的DNA中间体的能力。我们的研究结果表明，XRCC 1是植物碱基切除修复的一个组成部分，并在拟南芥主动DNA去甲基化过程中的几个阶段发挥作用。
DNA methylation patterns are the dynamic outcome of antagonist methylation and demethylation mechanisms, but the latter are still poorly understood. Active DNA demethylation in plants is mediated by a family of DNA glycosylases typified by Arabidopsis ROS1 (repressor of silencing 1). ROS1 and its homologs remove 5-methylcytosine and incise the sugar backbone at the abasic site, thus initiating a base excision repair pathway that finally inserts an unmethylated cytosine. The DNA 3'-phosphatase ZDP processes some of the incision products generated by ROS1, allowing subsequent DNA polymerization and ligation steps. In this work, we examined the possible role of plant XRCC1 (x-ray cross-complementing group protein 1) in DNA demethylation. We found that XRCC1 interacts in vitro with ROS1 and ZDP and stimulates the enzymatic activity of both proteins. Furthermore, extracts from xrcc1 mutant plants exhibit a reduced capacity to complete DNA demethylation initiated by ROS1. An anti-XRCC1 antibody inhibits removal of the blocking 3'-phosphate in the single-nucleotide gap generated during demethylation and reduces the capacity of Arabidopsis cell extracts to ligate a nicked DNA intermediate. Our results suggest that XRCC1 is a component of plant base excision repair and functions at several stages during active DNA demethylation in Arabidopsis.