Institute of Bioinformatics and Structural Biology, College of Life Science, National Tsing Hua University, Hsin-Chu, 30013, Taiwan, ROC.
Plant Mol Biol. 2010 Nov;74(4-5):453-66. doi: 10.1007/s11103-010-9685-5. Epub 2010 Sep 16.
The Ku heterodimer, a DNA repair protein complex consisting of 70- and 80-kDa subunits, is involved in the non-homologous end-joining (NHEJ) pathway. Plants are thought to use the NHEJ pathway primarily for the repair of DNA double-strand breaks (DSBs). The Ku70/80 protein has been identified in many plants and been shown to possess several similar functions to its counter protein complex in mammals. In the present study, ovate family protein 1 (AtOFP1) was demonstrated to be a plant Ku-interacting protein by yeast two-hybrid screening and the GST pull-down assay. Truncation analysis revealed that the C-terminal domain of AtKu70 contains interacting sites for AtOFP1. The electrophoretic mobility shift assay (EMSA) indicated that AtOFP1 is also a DNA binding protein with its binding domain at the N-terminus. In 3-week-old seedlings, expression of the AtOFP1 gene increased after exposure to DNA-damaging agents (such as methyl methanesulfonate (MMS) and menadione) in a time dependent manner. Seedlings lacking the AtOFP1 protein were more sensitive to MMS and menadione as compared with wild-type. Furthermore, similar to AtKu70(-/-) and AtKu80(-/-), the AtOFP1(-/-) mutant showed relatively lower NHEJ activity in vivo. Taken together, these results suggest that AtOFP1 may play a role in DNA repair through the NHEJ pathway accompanying with the AtKu protein.
Ku 异源二聚体是一种由 70kDa 和 80kDa 亚基组成的 DNA 修复蛋白复合物，参与非同源末端连接（NHEJ）途径。人们认为植物主要通过 NHEJ 途径修复 DNA 双链断裂（DSBs）。Ku70/80 蛋白已在许多植物中被鉴定出来，并被证明具有与其在哺乳动物中的对应蛋白复合物相似的几种功能。在本研究中，卵形家族蛋白 1（AtOFP1）通过酵母双杂交筛选和 GST 下拉测定被证明是一种植物 Ku 相互作用蛋白。截断分析表明，AtKu70 的 C 末端结构域包含与 AtOFP1 相互作用的位点。电泳迁移率变动分析（EMSA）表明，AtOFP1 也是一种 DNA 结合蛋白，其结合域位于 N 端。在 3 周龄幼苗中，AtOFP1 基因的表达在暴露于 DNA 损伤剂（如甲基甲磺酸酯（MMS）和维生素 K3）后随时间增加。与野生型相比，缺乏 AtOFP1 蛋白的幼苗对 MMS 和维生素 K3 更敏感。此外，与 AtKu70（-/-）和 AtKu80（-/-）相似，AtOFP1（-/-）突变体在体内的 NHEJ 活性相对较低。总之，这些结果表明 AtOFP1 可能通过与 AtKu 蛋白一起参与 NHEJ 途径在 DNA 修复中发挥作用。
