Mutational analysis of RAG1 and RAG2 identifies three catalytic amino acids in RAG1 critical for both cleavage steps of V(D)J recombination

Abstract

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.

Figure 1

Targets for mutagenesis. Alignments of the predicted protein sequences of RAG1 (nine species) and RAG2 (seven species) were performed with ClustalW and used to determine conserved amino acids for mutagenesis. Accession nos. for RAG1 sequences are as follows: murine (P15919), human (P15918), rabbit (P34088), opossum (U51897), chicken (P24271), Xenopus (L19324.1), bull shark (U62645.1), rainbow trout (U15663), and zebrafish (U71093). Accession nos. for RAG2 sequences are as follows: murine (M64796), human (P55895), rabbit (M99311), chicken (M58531), Xenopus (L19325), rainbow trout (U31670), and zebrafish (U71094.1). Conserved aspartic acids (D) and glutamic acids (E) are shown as white on a black background. Positions of conserved charge (D or E), are black on a gray background. Nonconserved positions also chosen for mutagenesis are shown as white on gray.

Figure 2

RAG2 does not contribute conserved acidic amino acid residues to cleavage. Signal joints formed in vivo were detected by PCR and Southern blotting. The expected size of the signal joint-containing product is 220 bp. (1×) A PCR reaction containing one-thirtieth of the DNA recovered from a single transfection; (1:10, 1:100) PCR reactions containing the indicated dilutions. All mutants were assayed at the 1× concentration. The marker lane contains a radiolabeled 1-kb ladder (GIBCO/BRL). (Open triangle) 12-RSS; (solid triangle) 23-RSS; (rectangles) coding segments; (arrows) PCR primers; (SJ) signal joint; (WT) wild-type tRAG-1/tRAG-2.

Figure 3

Several RAG1 mutants are severely impaired for cleavage. Signal ends formed in vivo were detected by ligation-mediated PCR for the 23 RSS. (Similar results were obtained in assays for the 12 RSS; data not shown.) (1X) Ligations containing one-fifteenth of the DNA recovered from a single transfection, diluted 1:100 then assayed by PCR. All mutants were assayed at the 1× concentration. The expected product for cleavage at the 23 RSS is 128 bp. (SE) Signal end; parallel lines denote ligation primers; other symbols are as in Fig. 2.

Figure 4

Binding of RAG1 mutants to a 12 RSS. DNA binding was assayed by electrophoretic mobility shift with a radiolabeled oligonucleotide probe containing a 12 RSS. Proteins tested in lanes 1–5 were MBP fusions (of wild-type or mutant tRAG1 and wild-type tRAG2). In lanes 6–14, the RAG proteins do not contain the MBP fusion; hence, the mobility of the DNA–protein complex is faster than observed in lanes 1–5. Some mutants (D600N, D708N) were tested both as MBP and non-MBP forms with the same results.

Figure 5

Class-II mutants are defective for both nicking and hairpin formation. Cleavage activity was tested in Mn²⁺ with the same protein preparations used in Fig. 4 with a radiolabeled oligonucleotide substrate containing a 12 RSS. The substrate is labeled on the 5′ end of the top strand, such that nicking produces a 16-nucleotide product and transesterification generates a 32-nucleotide hairpin. (A) A non-nicked substrate is used to assay nicking and hairpin formation. The species migrating slightly faster than the hairpin product (NS) does not depend on RAG-mediated cleavage (lane 13) and is a nonspecific band present in the substrate preparation. (B) A prenicked substrate is used to assay specifically for hairpin formation. (WT) Incubations with wild-type tRAG1 and tRAG2. (1:10) incubations done with a 1:10 dilution of wild-type tRAG1 and tRAG2. (R2 only) control incubations performed only with tRAG2.

Figure 5

Class-II mutants are defective for both nicking and hairpin formation. Cleavage activity was tested in Mn²⁺ with the same protein preparations used in Fig. 4 with a radiolabeled oligonucleotide substrate containing a 12 RSS. The substrate is labeled on the 5′ end of the top strand, such that nicking produces a 16-nucleotide product and transesterification generates a 32-nucleotide hairpin. (A) A non-nicked substrate is used to assay nicking and hairpin formation. The species migrating slightly faster than the hairpin product (NS) does not depend on RAG-mediated cleavage (lane 13) and is a nonspecific band present in the substrate preparation. (B) A prenicked substrate is used to assay specifically for hairpin formation. (WT) Incubations with wild-type tRAG1 and tRAG2. (1:10) incubations done with a 1:10 dilution of wild-type tRAG1 and tRAG2. (R2 only) control incubations performed only with tRAG2.

Figure 6

Class-I, but not class-II, mutants are capable of DSB formation. Crude extracts containing RAG proteins were assayed with a plasmid substrate in the presence of Mn²⁺. Cleavage products were visualized by Southern blotting. Lanes 13–17 represent a longer exposure of lanes 8–12. Substrate and expected cleavage products are illustrated at right.

Figure 7

Analysis of charge-conserving substitution mutants in vivo. Signal ends generated in vivo by the indicated RAG mutants were detected by ligation-mediated PCR as described in the legend to Fig. 3.

Figure 8

Mn²⁺ rescue of cysteine substitution mutants. The indicated mutants were tested in the crude extract system for their ability to catalyze cleavage of plasmid substrates in Mn²⁺ (left) and in Mg²⁺ (right). Substrate and expected cleavage products are illustrated at right.

Figure 9

Sequence alignments of catalytic residues. Amino acid sequence alignments are shown for the relevant regions of RAG-1 from several organisms (A) and for selected retroviral integrase superfamily members and murine RAG-1 (B). (Asterisks) The amino acids defined by catalytic-deficient (class II) mutants. Highlighted amino acids were grouped as follows according to Engelman and Craigie (1992): (G,A,S,T,P); (L,I,V,M); (F,Y,W); (D,E,N,Q); (K,R,H); C. Amino acid numbers correspond to murine RAG-1.