Applied Biology Section, Molecular Biology Division, Bhabha Atomic Research Centre, Mumbai, Maharashtra 400085, India; Homi Bhabha National Institute, Anushakti Nagar, Mumbai, Maharashtra 400094, India; Department of Chemical Sciences, Tata Institute of Fundamental Research, Homi Bhabha Road, Colaba, Mumbai, 400 005, India.
Applied Biology Section, Molecular Biology Division, Bhabha Atomic Research Centre, Mumbai, Maharashtra 400085, India; Homi Bhabha National Institute, Anushakti Nagar, Mumbai, Maharashtra 400094, India.
Plant Sci. 2021 Jan;302:110730. doi: 10.1016/j.plantsci.2020.110730. Epub 2020 Oct 23.
Chromosomal breaks occur in the genome of all living organisms upon exposure to ionizing radiation, xenobiotics and as intermediates during normal cell cycle progression. Most of the information on DNA repair process has emerged from bacteria, human, mice, and yeast while information on plant DNA repair genes and proteins is limited. Among other DNA repair proteins, MRE11 forms the core of the MRN (Mre11-Rad50-Nbs1) complex and is the first responder to double strand breaks (DSBs), promotes repair either by Non-Homologous End Joining (NHEJ) or Homologous Recombination (HR). Till date, MRE11 has not been biochemically characterized from plant systems. Here, we report the in vitro biochemical activities of Oryza sativa MRE11. We cloned and purified the N- terminal region of OsMre11, which represents both the nuclease and DNA binding domains. The N- terminal end of OsMre11-N protein (∼55.0 kDa) showed binding activity with dsDNA, ssDNA and G-quadruplex DNA. Tryptophan fluorescence analysis also showed that OsMre11-N protein binds to ssDNA, dsDNA and G4 DNA in a protein concentration dependant manner. Additionally, OsMre11 protein showed exonuclease activity only in the presence of Mn. A protein concentration dependant endonuclease activity also was observed and was enhanced in the presence of Mn, Mg and Ca. Put together, OsMre11 has properties similar to its counterparts in yeast and humans and may play an important role in cellular response to DNA damage in plants, especially rice.
染色体断裂在所有生物体的基因组中都会发生，当暴露于电离辐射、外源化学物质以及正常细胞周期进展过程中的中间体时。大多数关于 DNA 修复过程的信息都来自细菌、人类、老鼠和酵母，而关于植物 DNA 修复基因和蛋白质的信息则有限。在其他 DNA 修复蛋白中，MRE11 形成了 MRN（Mre11-Rad50-Nbs1）复合物的核心，是对双链断裂（DSBs）的第一反应者，通过非同源末端连接（NHEJ）或同源重组（HR）促进修复。迄今为止，MRE11 尚未从植物系统中进行生化表征。在这里，我们报告了拟南芥 MRE11 的体外生化活性。我们克隆并纯化了 OsMre11 的 N 端区域，它代表了核酸酶和 DNA 结合结构域。OsMre11-N 蛋白的 N 端（约 55.0 kDa）表现出与 dsDNA、ssDNA 和 G-四链体 DNA 的结合活性。色氨酸荧光分析还表明，OsMre11-N 蛋白以蛋白浓度依赖的方式结合 ssDNA、dsDNA 和 G4 DNA。此外，OsMre11 蛋白仅在 Mn 的存在下表现出外切核酸酶活性。还观察到一种蛋白浓度依赖的内切核酸酶活性，并且在 Mn、Mg 和 Ca 的存在下增强。综上所述，OsMre11 具有与其在酵母和人类中的对应物相似的特性，可能在植物，特别是水稻对 DNA 损伤的细胞反应中发挥重要作用。
