MCB
Home Help [Feedback] [For Subscribers] [Archive] [Search] [Contents]
This Article
Right arrow Full Text
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Gomez, C. A.
Right arrow Articles by Santagata, S.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Gomez, C. A.
Right arrow Articles by Santagata, S.

 Previous Article  |  Next Article 

Molecular and Cellular Biology, August 2000, p. 5653-5664, Vol. 20, No. 15
0270-7306/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

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

Carlos A. Gomez,1 Leon M. Ptaszek,1,Dagger Anna Villa,2 Fabio Bozzi,2 Cristina Sobacchi,2 Edward G. Brooks,3 Luigi D. Notarangelo,4 Eugenia Spanopoulou,1,§ Z. Q. Pan,1 Paolo Vezzoni,2 Patricia Cortes,5 and Sandro Santagata1,*

Ruttenberg Cancer Center1 and Immunobiology Center,5 Mount Sinai School of Medicine of New York University, New York, New York 10029; Department of Human Genome and Multifactorial Disease, Istituto di Tecnologie Biomediche Avanzate, Consiglio Nazionale delle Ricerche, 20090 Segrate, Milan,2 and Department of Paediatrics, Spedali Civili and University of Brescia, Brescia 25123,4 Italy; and University of Texas Medical Branch, Department of Pediatrics, Child Health Research Center, Galveston, Texas 77555-03663

Received 29 February 2000/Returned for modification 5 April 2000/Accepted 13 May 2000

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.

* Corresponding author. Mailing address: Ruttenberg Cancer Center, Mount Sinai School of Medicine, Box 1130, 1425 Madison Ave., New York, NY 10029. Phone: (212) 659-5525. Fax: (212) 849-2446. E-mail: santas01{at}doc.mssm.edu.

dagger Manuscript 41 of the Cariplo-ITBA project Genoma 2000, directed by R. Dulbecco and funded by Cariplo.

Dagger Present address: Section of Immunobiology, Yale University School of Medicine, New Haven, CT 06520.

§ Eugenia Spanopoulou was killed in the crash of Swiss Air flight 111 on 2 September 1998.

Molecular and Cellular Biology, August 2000, p. 5653-5664, Vol. 20, No. 15
0270-7306/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.


This article has been cited by other articles:



Home Help [Feedback] [For Subscribers] [Archive] [Search] [Contents]
J. Bacteriol. J. Virol. Eukaryot. Cell
Microbiol. Mol. Biol. Rev. Clin. Vaccine Immunol. All ASM Journals

Copyright © 2000 by the American Society for Microbiology. All rights reserved.