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Mutations of acidic residues in RAG1 define the active site of the V(D)J recombinase
Open accessAbstract
The RAG1 and RAG2 proteins collaborate to initiate V(D)J recombination by binding recombination signal sequences (RSSs) and making a double-strand break between the RSS and adjacent coding DNA. Like the reactions of their biochemical cousins, the bacterial transposases and retroviral integrases, cleavage by the RAG proteins requires a divalent metal ion but does not involve a covalent protein/DNA intermediate. In the transposase/integrase family, a triplet of acidic residues, commonly called a DDE motif, is often found to coordinate the metal ion used for catalysis. We show here that mutations in each of three acidic residues in RAG1 result in mutant derivatives that can bind the RSS but whose ability to catalyze either of the two chemical steps of V(D)J cleavage (nicking and hairpin formation) is severely impaired. Because both chemical steps are affected by the same mutations, a single active site appears responsible for both reactions. Two independent lines of evidence demonstrate that at least two of these acidic residues are directly involved in coordinating a divalent metal ion: The substitution of Cys for Asp allows rescue of some catalytic function, whereas an alanine substitution is no longer subject to iron-induced hydroxyl radical cleavage. Our results support a model in which the RAG1 protein contains the active site of the V(D)J recombinase and are interpreted in light of predictions about the structure of 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 [21]
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Sorted by citations
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BackgroundRAG1 Core and V(D)J Recombination Signal Sequences Were Derived from Transib Transposons
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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 S3).”
Background
Metadata
- Authors
- Kim, D. R., Dai, Y., Mundy, C. L., Yang, W., & Oettinger, M. A.
- Affiliations
- National Institutes of Health (US)
- Venue
- Genes & Development
- Publisher
- Cold Spring Harbor Laboratory
- Published
- 1999-12-01
- Volume
- 13
- Issue
- 23
- Pages
- 3070-3080
- ISSN
- 0890-9369 (ISSN-L)
- PMC
- PMC317176
- OpenAlex
- W1984676925
- Cited by
- 199
- References
- 52
- Citations by year
- 2024: 4 · 2023: 5 · 2022: 3 · 2021: 1 · 2020: 3 · 2019: 4 · 2018: 3 · 2017: 2 · 2016: 3 · 2015: 16 · 2014: 8 · 2013: 6 · 2012: 6
- Type
- posted content
- Language
- en
- Open access
- bronze
- doi.org
- Locations
- Genes & Development (publishedVersion)
- PubMed Central (publishedVersion)
- PubMed (publishedVersion)
- Funders
- Cancer Research Institute
- National Institutes of Health
- National Institute of General Medical Sciences (GM58026)
- 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 › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Genomics and Chromatin Dynamics
- Keywords
- RAG1, RAG2, V(D)J recombination, recombination signal sequences, acidic residues, active site, nicking, hairpin formation, immunoglobulin gene rearrangement
- MeSH terms
- Amino Acid Sequence, Animals, Aspartic Acid, Binding Sites, DNA Nucleotidyltransferases, DNA-Binding Proteins, HeLa Cells, Humans, Iron, Manganese, Molecular Sequence Data, Nuclear Proteins, Phenotype, Protein Binding, Recombinant Fusion Proteins, Structure-Activity Relationship, Gene Rearrangement, Mutagenesis, Site-Directed, Protein Structure, Secondary, Protein Structure, Tertiary, Hydroxyl Radical, Homeodomain Proteins, Amino Acid Substitution, Catalytic Domain, VDJ Recombinases, Mice
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