The DOI matches its Crossref record; 74 of the 74 references it cites were found in a registry or an index.
Mutational analysis of RAG1 and RAG2 identifies three catalytic amino acids in RAG1 critical for both cleavage steps of V(D)J recombination
Open accessAbstract
RAG1 and RAG2 initiate V(D)J recombination, the process of rearranging the antigen-binding domain of immunoglobulins and T-cell receptors, by introducing site-specific double-strand breaks (DSB) in chromosomal DNA during lymphocyte development. These breaks are generated in two steps, nicking of one strand (hydrolysis), followed by hairpin formation (transesterification). The nature and location of the RAG active site(s) have remained unknown. Because acidic amino acids have a critical role in catalyzing DNA cleavage by nucleases and recombinases that require divalent metal ions as cofactors, we hypothesized that acidic active site residues are likewise essential for RAG-mediated DNA cleavage. We altered each conserved acidic amino acid in RAG1 and RAG2 by site-directed mutagenesis, and examined >100 mutants using a combination of in vivo and in vitro analyses. No conserved acidic amino acids in RAG2 were critical for catalysis; three RAG1 mutants retained normal DNA binding, but were catalytically inactive for both nicking and hairpin formation. These data argue that one active site in RAG1 performs both steps of the cleavage reaction. Amino acid substitution experiments that changed the metal ion specificity suggest that at least one of these three residues contacts the metal ion(s) directly. These data suggest that RAG-mediated DNA cleavage involves coordination of divalent metal ion(s) by RAG1.
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“All three core residues from the catalytic DDE triad in the RAG1 proteins (residues 603, 711, and 965) that are necessary for V(D)J recombination [21,22] are conserved in the Transib TPases (Figures 3 and Figure S3).”
From RAG1 Core and V(D)J Recombination Signal Sequences Were Derived from Transib Transposons (2005) · cites this paper as [22]
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Self-citationMetal ion catalysis in the Tetrahymena ribozyme reaction
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Lieber, M. R. (2004) · Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature · doi:10.3410/f.1011614.213423
V(D)J recombination: mechanism and consequences
Gellert, M. F. (2007) · Topics in current genetics · doi:10.1007/978-3-540-71021-9_16
Faculty Opinions recommendation of Cracking the DNA code for V(D)J recombination.
Schatz, D. G. (2018) · Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature · doi:10.3410/f.733014924.793549792
RAG1: THE CORNERSTONE GENE INVOLVED IN VARIOUS IMMUNE DISEASES AND CANCERS
Fawzy, M., Nazmy, M., & Fathy, M. (2023) · Bulletin of Pharmaceutical Sciences Assiut · doi:10.21608/bfsa.2023.327556
The Role of RAG in V(D)J Recombination
Xiao, J., Martin, E. C., Wang, K., & Schatz, D. G. (2024) · Elsevier eBooks · doi:10.1016/b978-0-128-24465-4.00019-3
Zhang, R., Wu, Y., Sun, T., Luan, T., Ren, Y., Zhang, Y., LIN, L., Meng, T., Wang, J., & Qiu, Y. (2026) · Frontiers in Immunology · doi:10.3389/fimmu.2026.1757508
Les DDEs transposons à l’origine de l’innovation génétique : exemple du transposon RAG
Tsakou-Ngouafo, L. (2022) · theses.fr (ABES) · doi:10.70675/89cc8269ze49bz4eabz906dzf93f1457a05a
P Transposable Elements in Drosophila and other Eukaryotic Organisms
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Metadata
- Authors
- Landree, M. A., Wibbenmeyer, J. A., & Roth, D. B.
- Affiliations
- Baylor College of Medicine (US)
- Howard Hughes Medical Institute (US)
- Corresponding author
- Roth, D. B.
- ORCID
- Roth, D. B.
- Venue
- Genes & Development
- Publisher
- Cold Spring Harbor Laboratory
- Published
- 1999-12-01
- Volume
- 13
- Issue
- 23
- Pages
- 3059-3069
- ISSN
- 0890-9369 (ISSN-L)
- PMC
- PMC317185
- OpenAlex
- W2075932264
- Cited by
- 209
- References
- 73
- Citations by year
- 2026: 1 · 2025: 1 · 2024: 5 · 2023: 5 · 2022: 5 · 2021: 1 · 2020: 1 · 2019: 5 · 2018: 4 · 2017: 3 · 2016: 3 · 2015: 15 · 2014: 9 · 2013: 7 · 2012: 6
- Type
- posted content
- Language
- en
- Open access
- bronze
- doi.org
- Locations
- Genes & Development (publishedVersion)
- PubMed Central (publishedVersion)
- PubMed (publishedVersion)
- Funders
- National Institute of General Medical Sciences (GM08231, T32GM008231)
- National Institute of Allergy and Infectious Diseases (T32AI007495, AI07495, AI 36420, R01AI036420)
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › DNA Repair Mechanisms
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › CRISPR and Genetic Engineering
- Life Sciences › Immunology and Microbiology › Immunology › T-cell and B-cell Immunology
- Keywords
- RAG1, RAG2, V(D)J recombination, DNA double-strand breaks, nicking, hairpin formation, transesterification, catalytic site, acidic amino acids, site-directed mutagenesis, lymphocyte development
- MeSH terms
- Amino Acid Sequence, Animals, Binding Sites, Catalysis, Cricetulus, Cysteine, DNA, DNA Nucleotidyltransferases, DNA-Binding Proteins, Cricetinae, Humans, Lymphocytes, Manganese, Molecular Sequence Data, Nuclear Proteins, Recombinant Fusion Proteins, Structure-Activity Relationship, Transfection, Vertebrates, Gene Rearrangement, Mutagenesis, Site-Directed, Sequence Alignment, CHO Cells, Sequence Homology, Amino Acid, Homeodomain Proteins, Amino Acid Substitution, Catalytic Domain, VDJ Recombinases
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