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Vol. 13, No. 23, pp. 3059-3069, December 1, 1999
1 Program in Cell and Molecular Biology,
2 Department of Microbiology and Immunology, 3 Howard
Hughes Medical Institute, Baylor College of Medicine,
Houston, Texas 77030 USA
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.
[Key Words: RAG1; RAG2; V(D)J recombination; immunoglobulin]
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