Department of Molecular Medicine and Institute of Biotechnology, University of Texas Health Science Center, San Antonio, Texas, USA.
Mol Cell Biol. 2012 Sep;32(18):3663-80. doi: 10.1128/MCB.00406-12. Epub 2012 Jul 9.
RAD51 is important for restarting stalled replication forks and for repairing DNA double-strand breaks (DSBs) through a pathway called homology-directed repair (HDR). However, analysis of the consequences of specific RAD51 mutants has been difficult since they are toxic. Here we report on the dominant effects of two human RAD51 mutants defective for ATP binding (K133A) or ATP hydrolysis (K133R) expressed in mouse embryonic stem (ES) cells that also expressed normal mouse RAD51 from the other chromosome. These cells were defective for restarting stalled replication forks and repairing breaks. They were also hypersensitive to camptothecin, a genotoxin that generates breaks specifically at the replication fork. In addition, these cells exhibited a wide range of structural chromosomal changes that included multiple breakpoints within the same chromosome. Thus, ATP binding and hydrolysis are essential for chromosomal maintenance. Fusion of RAD51 to a fluorescent tag (enhanced green fluorescent protein [eGFP]) allowed visualization of these proteins at sites of replication and repair. We found very low levels of mutant protein present at these sites compared to normal protein, suggesting that low levels of mutant protein were sufficient for disruption of RAD51 activity and generation of chromosomal rearrangements.
RAD51 对于重新启动停滞的复制叉以及通过同源定向修复（HDR）途径修复 DNA 双链断裂（DSBs）非常重要。然而，由于 RAD51 突变体具有毒性，因此对其特定突变体的后果进行分析一直很困难。在这里，我们报告了在表达正常小鼠 RAD51 的另一条染色体的情况下，两种人 RAD51 突变体（ATP 结合缺陷的 K133A 或 ATP 水解缺陷的 K133R）在小鼠胚胎干细胞（ES 细胞）中的显性效应。这些细胞无法重新启动停滞的复制叉，也无法修复断裂。它们对喜树碱（一种在复制叉处产生断裂的遗传毒素）也非常敏感。此外，这些细胞表现出广泛的结构染色体变化，包括同一染色体内的多个断裂点。因此，ATP 结合和水解对于染色体维持是必不可少的。RAD51 与荧光标签（增强型绿色荧光蛋白[eGFP]）融合，使我们能够在复制和修复部位可视化这些蛋白质。与正常蛋白相比，我们发现这些部位的突变蛋白水平非常低，这表明低水平的突变蛋白足以破坏 RAD51 活性并产生染色体重排。
