Mutations in conserved regions of the predicted RAG2 kelch repeats block initiation of V(D)J recombination and result in primary immunodeficiencies

The V(D)J recombination reaction is composed of multiple nucleolytic proces sing 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 w ithin and between kelch repeat-containing proteins. Here we demonstrate tha t 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. Gre en 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, theref ore implicating a defect in the early steps of the recombination reaction a s the basis of the clinical phenotype. The present experiments, performed w ith an extensive panel of site-directed mutations within each of the six ke lch motifs, further support the critical role of both hydrophobic and glyci ne-rich regions within the second beta-strand for RAG1-RAG2 interaction and recombination signal recognition and cleavage. In contrast, multiple mutat ions 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 kel ch repeats for V(D)J recombination and highlight the importance of the cons erved elements of the kelch motif.