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. 1999 Dec 1;13(23):3070-80.
doi: 10.1101/gad.13.23.3070.

Mutations of acidic residues in RAG1 define the active site of the V(D)J recombinase

D R Kim  1 , Y Dai, C L Mundy, W Yang, M A Oettinger
Affiliations

Mutations of acidic residues in RAG1 define the active site of the V(D)J recombinase

D R Kim et al. Genes Dev. .

Abstract

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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Figures

Figure 1
Figure 1
Iron-induced hydroxyl radical cleavage of RAG1. (A) Diagram of the MBP–RAG1 fusion protein used for this analysis, with positions of the MBP and 8 histidine tag indicated. (B) Western blot with anti-MBP antibody reveals a ∼70-kD iron-dependent cleavage product. (C) Western blot with anti-His tag antibody reveals a ∼47-kD carboxy-terminal cleavage product. The position of the cleaved products is indicated by an arrow; the presence (+) or absence (−) of Fe2+ in the reaction is indicated.
Figure 2
Figure 2
RAG1 mutant proteins D600A, D708A, and E962A are defective in V(D)J cleavage. Single site cleavage of a 32P-labeled 12-RSS oligonucleotide substrate (see Materials and Methods) is shown. Lane 1 contains no RAG protein; all other lanes contain the indicated RAG1 protein in addition to RAG2. Reactions were carried out in 0.1 mm Mn2+ for 30 min. The positions of the substrate (S) and nicked (N) and hairpin (HP) cleavage products are marked.
Figure 3
Figure 3
DNA binding and synaptic complex formation. (A) Bandshift assays in the presence of a labeled 12 RSS are shown for the indicated RAG1 derivatives. The presence (+) or absence (−) of RAG1 and RAG2 in the reactions is indicated. (B) Bandshifts assays were carried out as in A with the addition of HMG1 and an unlabeled 23-RSS oligonucleotide (see Materials and Methods; Hiom and Gellert 1998). The presence (+) or absence (−) of the 23 RSS is noted. Lanes 3–20 contain RAG2 protein. The positions of the RAG1 alone (R1), single complex (SC1 and SC2), and paired complex (PC) bands, as well as the position of the labeled substrate (S), are indicated.
Figure 4
Figure 4
Effects of RAG1 mutations on coupled cleavage and hairpin formation. (A) Coupled cleavage of a tethered substrate by the indicated RAG1 derivatives in reaction buffer containing Mn2+ or Mg2+ is shown. The substrate (S) is labeled at the 23 RSS and the positions of cleavage products with a hairpin only at the 23 RSS (23H), a hairpin at the 23 RSS, and a nick at the 12 RSS (23H12N) or a hairpin at both signals (23H12H) are marked. (B) Hairpin formation on a prenicked substrate following a 2-hr incubation is shown for either the wild-type RAG1 protein or the indicated mutant derivatives. The presence of increasing concentrations of Mn2+ (0.05, 0.2, 2, 10 mm) is indicated by an open wedge.
Figure 5
Figure 5
Effects of RAG1 mutation on transposition. Transposition of 32P-labeled precleaved 12/23 RSS donors into a target plasmid is shown for wild-type (WT) and mutant RAG1 proteins in the presence of the indicated divalent metal ion. Arrows indicate the position of linearized and nicked target DNA. Lanes 14–20 come from the same exposure of the same gel, spliced to remove intervening lanes.
Figure 6
Figure 6
The D600A/D708A double mutant is not subject to iron-induced cleavage. (A) Cleavage products were visualized with anti-MBP antibodies. Cleavage reactions were carried out in the absence of metal (−) or in the presence of iron (Fe2+) or calcium (Ca2+). (B) Carboxy-terminal cleavage products of RAG1 derivatives were visualized with anti-histidine tag antibody. The presence (+) or absence (−) of Fe2+ is indicated. (WT) Wild-type RAG1.
Figure 7
Figure 7
Predicted structure of the core domain of RAG1. The structure predicted by the PSIpred program for RAG1 amino acids 481–1020 is shown above the murine RAG1 sequence. (H) Helix; (C) coil; (E) sheet. Structural predictions with a high confidence level (7–10) are indicated with uppercase letters; lower confidence levels are shown in lowercase. RAG1 sequences conserved between humans, mouse, chicken, Xenopus, and salmon are shown in uppercase letters; nonconserved residues in lowercase. Positions where either a D or E is observed are underlined. The positions of all residues mutagenized for this study are indicated with a black diamond; arrows and boldface type indicate the position of D600, D708, and E962.
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