RAD51 loss of function abolishes gene targeting and de-represses illegitimate integration in the moss Physcomitrella patens
RAD51 loss of function abolishes gene targeting and de-represses illegitimate integration in the moss Physcomitrella patens
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DOI：
10.1016/j.dnarep.2010.02.001
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发表时间：
2010-05-04
期刊：
影响因子：
3.8
通讯作者：
Nogue, F.
中科院分区：
文献类型：
--
作者：
Schaefer, D. G.;Delacote, F.;Nogue, F.
基因靶向（Gene targeting， GT）是一种重要的基础研究和应用研究工具，它将与染色体靶标具有序列同源性的转化DNA通过同源重组（homologous recombination， HR）整合到相应的位点。在真核生物中，GT从hr介导的双链断裂修复途径中招募酶。已经描述了不同的HR机制，这些机制依赖于Rad52上位基因群，但细胞对GT使用的具体机制尚不清楚。在酿酒酵母中，RAD52蛋白对GT至关重要，RAD51蛋白起次要作用。而在丝状真菌和动物细胞中，GT依赖于RAD51，受RAD52抑制的影响较弱。遗传证据还表明，DSB修复的非同源末端连接途径对GT效率有负面影响，但如何控制这两种途径之间的平衡尚不清楚。在这里，我们研究了RAD51在唯一一种具有高GT频率的植物——模式苔藓植物小壶藻（physcomitella patens）中的作用。我们的研究结果表明，这两个RAD51蛋白在维持基因组完整性和抵抗电离辐射方面具有部分冗余功能。此外，我们证明了两个RAD51蛋白功能的丧失完全消除了GT，并大大增加了这种苔藓的非法整合率。这些发现首次在植物中证实了RAD51在控制转基因过程中观察到的靶向整合和随机整合之间的平衡方面的关键作用，并证实了P. patens是研究高等真核生物GT的一个特别有趣的工具。(C) 2010 Elsevier B.V.版权所有
Gene targeting (GT) is a major tool for basic and applied research during which the transforming DNA, which shares sequence homology with a chromosomal target, integrates at the corresponding locus by homologous recombination (HR). In eukaryotes, GT recruits enzymes from the HR-mediated double strand break repair pathway. Different mechanisms of HR have been described which depend on the Rad52 epistasis group of genes, but which specific mechanism is used by the cell for GT remains unclear. In Saccharomyces cerevisiae, the RAD52 protein is essential for GT, and the RAD51 protein plays a minor role. In filamentous fungi and animal cells, however, GT depends on RAD51 and is weakly affected by suppression of RAD52. Genetic evidence also indicates that the non-homologous end-joining pathway of DSB repair has a negative impact on GT efficiencies, but how the balance between these two pathways is controlled is poorly understood. Here, we have examined the role of RAD51 in the only plant that exhibits high GT frequencies, the model bryophyte Physcomitrella patens. Our results show that the two RAD51 proteins have partially redundant functions in the maintenance of genome integrity and resistance to ionizing radiation. Furthermore, we demonstrate that loss of function of the two RAD51 proteins completely abolishes GT and strongly increases illegitimate integration rates in this moss. These findings demonstrate for the first time in plant the critical role of RAD51 in controlling the balance between targeted and random integration events observed upon transgenesis, and confirm that P. patens is a particularly interesting tool for studying GT in higher eukaryotes. (C) 2010 Elsevier B.V. All rights reserved.