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

Li, Wenhui and Chang, Fu-Chung and Desiderio, Stephen (2001) RAG-1 Mutations Associated with B-Cell-Negative SCID Dissociate the Nicking and Transesterification Steps of V(D)J Recombination. Molecular and Cellular Biology, 21 (12). pp. 3935-3946. ISSN 0270-7306. doi:10.1128/MCB.21.12.3935-3946.2001.

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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.

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Additional Information:Copyright © 2001, American Society for Microbiology. Received 13 November 2000/Returned for modification 9 January 2001/Accepted 30 March 2001 We thank Joanne Hesse, Dik van Gent, and Martin Gellert for reagents and the Howard Hughes Medical Institute Biopolymers Facility at Johns Hopkins for oligonucleotides. We are grateful to Patrick Swanson, Jinhak Lee, Amit Golding, Ashley Ross, and our colleagues in the Department of Molecular Biology and Genetics for stimulating discussions. Nick Dordai provided expert technical assistance. This work was supported by grant CA16519 from the National Cancer Institute and by the Howard Hughes Medical Institute.
Issue or Number:12
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ID Code:2753
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Deposited On:25 Apr 2006
Last Modified:08 Nov 2021 19:50

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