Dale A. Ramsden

Cleavage at a V(D)J recombination signal requires only RAG1 and RAG2 proteins and occurs in two steps

Formation of double-strand breaks at recombination signal sequences is an early step in V(D)J recombination. Here we show that purified RAG1 and RAG2 proteins are sufficient to carry out this reaction. The cleavage reaction can be divided into two distinct steps. First, a nick is introduced at the 5 end of the signal sequence. The other strand is then broken, resulting in a hairpin structure at the coding end and a blunt, 5-phosphorylated signal end. The hairpin is made as a direct consequence of the cleavage mechanism. Nicking and hairpin formation each require the presence of a signal sequence and both RAG proteins.

Ku protein stimulates DNA end joining by mammalian DNA ligases: a direct role for Ku in repair of DNA double‐strand breaks

Ku protein binds to DNA ends and is a cofactor for the DNA‐dependent protein kinase. Both of these components are involved in DNA double‐strand break repair, but it has not been clear if they function indirectly, by sensing DNA damage and activating other factors, or if they are more directly involved in the processing and rejoining of DNA breaks. We demonstrate that intermolecular ligation of DNA fragments is highly dependent on Ku under conditions designed to mimic those existing in the cell. This effect of Ku is specific to eukaryotic DNA ligases. Ku protein, therefore, has an activity consistent with a direct role in rejoining DNA breaks and independent of DNA‐dependent protein kinase.

Initiation of V(D)J recombination in a cell-free system

Cells performing V(D)J recombination make specific cuts in DNA at recombination signal sequences. Here, we show that nuclear extracts of pre-B cell lines carry out this specific cleavage. The products of cleavage are the same as found previously in thymocytes: full-length, blunt, 5-phosphorylated signal ends, and covalently sealed (hairpin) coding ends. A complete signal sequence is required. Recombinant RAG1 protein greatly increases activity and complements an inactive extract from a RAG1 (-/-) pre-B cell line. When the extracts are fractionated, cleavage activity correlates with the presence of RAG2 protein. These results suggest that RAG1 and RAG2 are components of the V(D)J recombinase.

The RAG1 and RAG2 Proteins Establish the 12/23 Rule in V(D)J Recombination

V(D)J recombination requires a pair of signal sequences with spacer lengths of 12 and 23 base pairs. Cleavage by the RAG1 AND RAG2 proteins was previously shown to demand only a single signal sequence. Here, we established conditions where 12- and 23-spacer signal sequences are both necessary for cleavage. Coupled cutting at both sites requires only the RAG1 and RAG2 proteins, but depends on the metal ion. In Mn2+, a single signal sequence supports efficient double strand cleavage, but cutting in Mg2+ requires two signal sequences and is best with the canonical 12/23 pair. Thus, the RAG proteins determine both aspects of the specificity of V(D)J recombination, the recognition of a single signal sequence and the correct 12/23 coupling in a pair of signals.

Distinct DNA sequence and structure requirements for the two steps of V(D)J recombination signal cleavage.

Cleavage of V(D)J recombination signals by purified RAG1 and RAG2 proteins permits the dissection of DNA structure and sequence requirements. The two recognition elements of a signal (nonamer and heptamer) are used differently, and their cooperation depends on correct helical phasing. The nonamer is most important for initial binding, while efficient nicking and hairpin formation require the heptamer sequence. Both nicking and hairpin formation are remarkably tolerant of variations in DNA structure. Certain flanking sequences inhibit hairpin formation, but this can be bypassed by base unpairing, and even a completely single‐stranded signal sequence is well utilized. We suggest that DNA unpairing around the signal‐coding border is essential for the initiation of V(D)J combination.

Cell-free V(D)J recombination.

V(D)J recombination generates diversity in the immune system through the lymphoid-specific assembly of multiple gene segments into functional immunoglobulin and T-cell receptor genes (for reviews, see refs 1, 2). The first step in V(D)J recombination is cleavage of DNA at recombination signal sequences. Cleavage produces a blunt DNA end on each signal sequence and a hairpin end on adjacent coding gene segments, and can be reproduced in vitro by using purified RAG1 and RAG2 proteins. The later steps involve processing and joining of the cleaved DNA ends, and until now have been studied only in cells. Here we reconstitute the complete V(D)J recombination reaction in a cell-free system. We find that the RAG proteins are not only involved in cleavage, but are also needed in the later steps for efficient joining of coding ends. Joining is largely directed by short pieces of identical sequence in the coding flanks, but addition of human DNA ligase I results in greater diversity. Coding junctions contain short deletions as well as additions complementary to a coding flank (P nucleotides). Addition of non-templated nucleotides into coding junctions is mediated by terminal deoxyribonucleotidyl transferase. The cell-free reaction can therefore reproduce the complete set of processing events that occur in cells.

SPECIFICITY IN V(D)J RECOMBINATION : NEW LESSONS FROM BIOCHEMISTRY AND GENETICS

Recent in vitro work on V(D)J recombination has helped to clarify its mechanism. The first stage of the reaction, which can be reproduced with the purified RAG1 and RAG2 proteins, is a site-specific cleavage that generates the same broken DNA species found in vivo. The cleavage reaction is closely related to known types of transpositional recombination, such as that of HIV integrase. All the site specificity of V(D)J recombination, including the 12/23 rule, is determined by the RAG proteins. The later steps largely overlap with the repair of radiation-induced DNA double-strand breaks, as indicated by the identity of several newly characterized factors involved in repair. These developments open the way for a thorough biochemical study of V(D)J recombination.

Initiation of V(D)J Recombination in a Cell-Free System by RAG1 and RAG2 Proteins

Mature T cell receptor (TCR) and immunoglobulin (Ig) genes are assembled from separate gene segments, which are flanked by recombination signal sequences (RSS), consisting of conserved heptamer and nonamer motifs separated by a spacer region of 12 or 23 base pairs (bp). V(D)J recombination results in a precise head-to-head ligation of two signal sequences in a signal joint, and the imprecise joining of two coding segments in a coding joint, which may contain additions or deletions of a few bp. The imprecise nature of coding joints led to the hypothesis that double strand breaks (DSB) might be intermediates in the recombination pathway These DSB can then be processed by an exonuclease and/or by terminal deoxynucleotidyl transferase (TdT) before formation of a coding joint.