Mutations in conserved regions of the predicted RAG2 kelch repeats block initiation of V(D)J recombination and result in primary immunodeficiencies
Mutations in conserved regions of the predicted RAG2 kelch repeats block initiation of V(D)J recombination and result in primary immunodeficiencies
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DOI：
10.1128/mcb.20.15.5653-5664.2000
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发表时间：
2000-08-01
影响因子：
5.3
通讯作者：
Santagata, S
中科院分区：
文献类型：
--
作者：
Gomez, CA;Ptaszek, LM;Santagata, S
V(D)J重组反应由重组激活蛋白RAG1和RAG2介导的多个核溶解处理步骤组成。序列分析表明，RAG2包含六个kelch重复基序，预计它们将形成一个六叶片的Beta螺旋桨结构，每个重复的第二个Beta链在含有kelch重复的蛋白质内部和之间都显示出明显的保守性。在这里，我们证明了RAG2重复序列2和5的预测第二β链内的G95R和Delta I273突变导致患者P1和P2的免疫缺陷。绿色荧光蛋白与突变蛋白的融合显示了适当的细胞核定位。然而，这两个突变都降低了RAG2与RAG1相互作用的能力，并阻止了重组信号的切割，因此暗示作为临床表型基础的重组反应的早期步骤存在缺陷。本实验在六个海带基序中的每个基序中进行了一组广泛的定点突变，进一步支持了第二β链中疏水区域和富含甘氨酸的区域对RAG1-RAG2相互作用以及重组信号识别和切割的关键作用。相反，kelch重复序列可变环区域内的多个突变对RAG1-RAG2相互作用有轻微影响或没有影响，因此对介导重组的能力也有影响。总之，这些数据证明了RAG2海带重复对V(D)J重组的关键作用，并突出了海带基序保守元件的重要性。
The V(D)J recombination reaction is composed of multiple nucleolytic processing steps mediated by the recombination-activating proteins RAG1 and RAG2. Sequence analysis has suggested that RAG2 contains six kelch repeat motifs that are predicted to form a six-bladed beta-propeller structure, with the second beta-strand of each repeat demonstrating marked conservation both within and between kelch repeat-containing proteins. Here we demonstrate that mutations G95R and Delta I273 within the predicted second beta-strand of repeats 2 and 5 of RAG2 lead to immunodeficiency in patients P1 and P2. Green fluorescent protein fusions with the mutant proteins reveal appropriate localization to the nucleus. However, both mutations reduce the capacity of RAG2 to interact with RAG1 and block recombination signal cleavage, therefore implicating a defect in the early steps of the recombination reaction as the basis of the clinical phenotype. The present experiments, performed with an extensive panel of site-directed mutations within each of the six kelch motifs, further support the critical role of both hydrophobic and glycine-rich regions within the second beta-strand for RAG1-RAG2 interaction and recombination signal recognition and cleavage. In contrast, multiple mutations within the variable-loop regions of the kelch repeats had either mild or no effects on RAG1-RAG2 interaction and hence on the ability to mediate recombination. In all, the data demonstrate a critical role of the RAG2 kelch repeats for V(D)J recombination and highlight the importance of the conserved elements of the kelch motif.