Boisvert François-Michel, Rhie Alexandre, Richard Stéphane, Doherty Aidan J
Terry Fox Molecular Oncology, Bloomfield Center for Research on Aging, Lady Davis Institute for Medical Research, Department of Oncology, McGill University, Montréal, Québec, Canada.
Cell Cycle. 2005 Dec;4(12):1834-41. doi: 10.4161/cc.4.12.2250. Epub 2005 Dec 14.
The p53-binding protein 1 (53BP1) is rapidly recruited to sites of DNA double-strand breaks and forms characteristics nuclear foci, demonstrating its role in the early events of detection, signaling and repair of damaged DNA. 53BP1 contains a glycine arginine rich (GAR) motif of unknown function within its kinetochore-binding domain. Herein, we show that the GAR motif of 53BP1 is arginine methylated by protein arginine methyltransferase 1 (PRMT1), the same methyltransferase that methylates MRE11. 53BP1 contains asymmetric dimethylarginines (aDMA) within cells, as detected with methylarginine-specific antibodies. Amino acid substitution of the arginines within the GAR motif of 53BP1 abrogated binding to single and double-stranded DNA, demonstrating that the GAR motif is required for DNA binding activity of 53BP1. Fibroblast cells treated with methylase inhibitors failed to relocalize 53BP1 to sites of DNA damage and formed few gamma-H2AX foci, consistent with our previous data that MRE11 fails to relocalize to DNA damage sites in cells treated with methylase inhibitors. Our findings identify the GAR motif as a region required for 53BP1 DNA binding activity and as the site of methylation by PRMT1.
p53结合蛋白1（53BP1）能迅速被招募到DNA双链断裂位点并形成特征性核灶，这表明它在受损DNA的检测、信号传导及修复的早期事件中发挥作用。53BP1在其动粒结合结构域内含有一个功能未知的富含甘氨酸精氨酸（GAR）的基序。在此，我们表明53BP1的GAR基序被蛋白精氨酸甲基转移酶1（PRMT1）甲基化，PRMT1也是使MRE11甲基化的同一种甲基转移酶。用甲基精氨酸特异性抗体检测发现，53BP1在细胞内含有不对称二甲基精氨酸（aDMA）。53BP1的GAR基序内精氨酸的氨基酸替换消除了其与单链和双链DNA的结合，这表明GAR基序是53BP1 DNA结合活性所必需的。用甲基化酶抑制剂处理的成纤维细胞未能将53BP1重新定位到DNA损伤位点，且形成的γ-H2AX灶很少，这与我们之前的研究数据一致，即在用甲基化酶抑制剂处理的细胞中，MRE11未能重新定位到DNA损伤位点。我们的研究结果确定GAR基序是53BP1 DNA结合活性所需的区域，也是PRMT1甲基化的位点。
