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 Li, W.
Right arrow Articles by Desiderio, S.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Li, W.
Right arrow Articles by Desiderio, S.

 Previous Article  |  Next Article 

Molecular and Cellular Biology, June 2001, p. 3935-3946, Vol. 21, No. 12
0270-7306/01/$04.00+0   DOI: 10.1128/MCB.21.12.3935-3946.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

RAG-1 Mutations Associated with B-Cell-Negative SCID Dissociate the Nicking and Transesterification Steps of V(D)J Recombination

Wenhui Li,dagger Fu-Chung Chang, and Stephen Desiderio*

Department of Molecular Biology and Genetics and Howard Hughes Medical Institute, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205

Received 13 November 2000/Returned for modification 9 January 2001/Accepted 30 March 2001

Some patients with B-cell-negative severe combined immune deficiency (SCID) carry mutations in RAG-1 or RAG-2 that impair V(D)J recombination. Two recessive RAG-1 mutations responsible for B-cell-negative SCID, R621H and E719K, impair V(D)J recombination without affecting formation of single-site recombination signal sequence complexes, specific DNA contacts, or perturbation of DNA structure at the heptamer-coding junction. The E719K mutation impairs DNA cleavage by the RAG complex, with a greater effect on nicking than on transesterification; a conservative glutamine substitution exhibits a similar effect. When cysteine is substituted for E719, RAG-1 activity is enhanced in Mn2+ but remains impaired in Mg2+, suggesting an interaction between this residue and an essential metal ion. The R621H mutation partially impairs nicking, with little effect on transesterification. The residual nicking activity of the R621H mutant is reduced at least 10-fold upon a change from pH 7.0 to pH 8.4. Site-specific nicking is severely impaired by an alanine substitution at R621 but is spared by substitution with lysine. These observations are consistent with involvement of a positively charged residue at position 621 in the nicking step of the RAG-mediated cleavage reaction. Our data provide a mechanistic explanation for one form of hereditary SCID. Moreover, while RAG-1 is directly involved in catalysis of both nicking and transesterification, our observations indicate that these two steps have distinct catalytic requirements.

* Corresponding author. Mailing address: Department of Molecular Biology and Genetics and Howard Hughes Medical Institute, The Johns Hopkins University School of Medicine, Baltimore, MD 21205. Phone: (410) 955-4735. Fax: (410) 955-9124. E-mail: sdesider{at}jhmi.edu.

dagger Present address: Division of Biology, California Institute of Technology, Pasadena, CA 91125.

Molecular and Cellular Biology, June 2001, p. 3935-3946, Vol. 21, No. 12
0270-7306/01/$04.00+0   DOI: 10.1128/MCB.21.12.3935-3946.2001
Copyright © 2001, 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 © 2001 by the American Society for Microbiology. All rights reserved.