Takata M, Sasaki M S, Tachiiri S, Fukushima T, Sonoda E, Schild D, Thompson L H, Takeda S
CREST Research Project, Radiation Genetics, Faculty of Medicine, Kyoto University, Sakyo-ku, Kyoto 606-8501, Japan.
Mol Cell Biol. 2001 Apr;21(8):2858-66. doi: 10.1128/MCB.21.8.2858-2866.2001.
The Rad51 protein, a eukaryotic homologue of Escherichia coli RecA, plays a central role in both mitotic and meiotic homologous DNA recombination (HR) in Saccharomyces cerevisiae and is essential for the proliferation of vertebrate cells. Five vertebrate genes, RAD51B, -C, and -D and XRCC2 and -3, are implicated in HR on the basis of their sequence similarity to Rad51 (Rad51 paralogs). We generated mutants deficient in each of these proteins in the chicken B-lymphocyte DT40 cell line and report here the comparison of four new mutants and their complemented derivatives with our previously reported rad51b mutant. The Rad51 paralog mutations all impair HR, as measured by targeted integration and sister chromatid exchange. Remarkably, the mutant cell lines all exhibit very similar phenotypes: spontaneous chromosomal aberrations, high sensitivity to killing by cross-linking agents (mitomycin C and cisplatin), mild sensitivity to gamma rays, and significantly attenuated Rad51 focus formation during recombinational repair after exposure to gamma rays. Moreover, all mutants show partial correction of resistance to DNA damage by overexpression of human Rad51. We conclude that the Rad51 paralogs participate in repair as a functional unit that facilitates the action of Rad51 in HR.
Rad51蛋白是大肠杆菌RecA的真核同源物，在酿酒酵母的有丝分裂和减数分裂同源DNA重组（HR）中均发挥核心作用，对脊椎动物细胞的增殖至关重要。基于与Rad51的序列相似性（Rad51旁系同源物），五个脊椎动物基因RAD51B、-C、-D以及XRCC2和-3与HR相关。我们在鸡B淋巴细胞DT40细胞系中生成了这些蛋白各自缺失的突变体，并在此报告四个新突变体及其互补衍生物与我们之前报道的rad51b突变体的比较。通过靶向整合和姐妹染色单体交换测定，Rad51旁系同源物突变均损害HR。值得注意的是，突变细胞系均表现出非常相似的表型：自发染色体畸变、对交联剂（丝裂霉素C和顺铂）杀伤高度敏感、对γ射线轻度敏感，以及在暴露于γ射线后的重组修复过程中Rad51焦点形成明显减弱。此外，所有突变体通过人Rad51的过表达均显示出对DNA损伤抗性的部分校正。我们得出结论，Rad51旁系同源物作为一个功能单元参与修复，促进Rad51在HR中的作用。
