Campbell C, Romero D P
Department of Pharmacology, Medical School, University of Minnesota, 3-249 Millard Hall, 435 Delaware Street SE, Minneapolis, MN 55455, USA.
Nucleic Acids Res. 1998 Jul 1;26(13):3165-72. doi: 10.1093/nar/26.13.3165.
The RAD51 gene is a eukaryotic homolog of rec A, a critical component in homologous recombination and DNA repair pathways in Escherichia coli . We have cloned the RAD51 homolog from Tetrahymena thermophila , a ciliated protozoan. Tetrahymena thermophila RAD51 encodes a 36.3 kDa protein whose amino acid sequence is highly similar to representative Rad51 homologs from other eukaryotic taxa. Recombinant Rad51 protein was purified to near homogeneity following overproduction in a bacterial expression system. The purified protein binds to both single- and double-stranded DNA, possesses a DNA-dependent ATPase activity and promotes intermolecular ligation of linearized plasmid DNA. While steady-state levels of Rad51 mRNA are low in normally growing cells, treatment with UV light resulted in a >100-fold increase in mRNA levels. This increase in mRNA was time dependent, but relatively independent of UV dose over a range of 1400-5200 J/m2. Western blot analysis confirmed that Rad51 protein levels increase upon UV irradiation. Exposure to the alkylating agent methyl methane sulfonate also resulted in substantially elevated Rad51 protein levels in treated cells, with pronounced localization in the macronucleus. These data are consistent with the hypothesis that ciliates such as T.thermophila utilize a Rad51-dependent pathway to repair damaged DNA.
RAD51基因是rec A的真核同源物，rec A是大肠杆菌同源重组和DNA修复途径中的关键成分。我们已经从纤毛原生动物嗜热四膜虫中克隆出了RAD51同源物。嗜热四膜虫RAD51编码一种36.3 kDa的蛋白质，其氨基酸序列与其他真核生物类群的代表性Rad51同源物高度相似。在细菌表达系统中过量表达后，重组Rad51蛋白被纯化至接近均一。纯化后的蛋白可与单链和双链DNA结合，具有依赖于DNA的ATP酶活性，并能促进线性化质粒DNA的分子间连接。虽然在正常生长的细胞中Rad51 mRNA的稳态水平较低，但紫外线处理导致mRNA水平增加了100倍以上。这种mRNA的增加是时间依赖性的，但在1400 - 5200 J/m2的范围内相对独立于紫外线剂量。蛋白质印迹分析证实，紫外线照射后Rad51蛋白水平增加。暴露于烷基化剂甲磺酸甲酯也导致处理细胞中Rad51蛋白水平大幅升高，在大核中有明显定位。这些数据与嗜热四膜虫等纤毛虫利用依赖Rad51的途径修复受损DNA这一假说一致。
