Mutations in Conserved Regions of the Predicted RAG2 Kelch Repeats Block Initiation of V(D)J Recombination and Result in Primary Immunodeficiencies

Abstract

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 β-propeller structure, with the second β-strand of each repeat demonstrating marked conservation both within and between kelch repeat-containing proteins. Here we demonstrate that mutations G95R and ΔI273 within the predicted second β-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 β-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.

ACKNOWLEDGMENTS

Carlos A. Gomez, Leon M. Ptaszek, and Anna Villa contributed equally to this work.

This work was supported by National Institutes of Health (NIH) grant AI40191 and a Cancer Research Institute Investigator Award to Z.Q.P., by Telethon grant E0917 to A.V., by NIH grant AI45996-02 and by a Cancer Research Institute Investigator Award to P.C., and by a predoctoral Training Grant in Cancer Biology from the NIH to S.S. Confocal laser scanning microscopy was performed under the guidance of Scott Henderson at the MSSM-CLSM core facility, supported with funding from NIH shared instrumentation grant 1 S10 RR0 9145-01 and NSF Major Research Instrumentation grant DBI-9724504. A.V. is the recipient of an AIRC travel fellowship for international scientific exchange.

We thank Anindita Bhoumik for assistance with subcloning. S.S. thanks Stuart Aaronson for guidance and support. L.M.P. is grateful to David G. Schatz for generous support and encouragement. We also thank David G. Schatz for comments on the manuscript.

ADDENDUM IN PROOF

A recent paper by Corneo et al. (B. Corneo, D. Moshous, I. Callebaut, R. de Chasseval, A. Fischer, and J. P. de Villartay, J. Biol. Chem. 275:12672–12675, 2000) describes three new mutations within RAG2 and provides support for the β-propeller model of RAG2 based on the clustering of mutations on one face of the molecule.