V(D)J recombination assembles immunoglobulin and T cell receptor genes during lymphocyte development through a series of carefully orchestrated DNA breakage and rejoining events. DNA cleavage requires a series of protein-DNA complexes containing the RAG1 and RAG2 proteins and recombination signals that flank the recombining gene segments. In this review, we discuss recent advances in our understanding of the function and domain organization of the RAG proteins, the composition and structure of RAG-DNA complexes, and the pathways that lead to the formation of these complexes. We also consider the functional significance of RAG-mediated histone recognition and ubiquitin ligase activities, and the role played by RAG in ensuring proper repair of DNA breaks made during V(D)J recombination. Finally, we propose a model for the formation of RAG-DNA complexes that involves anchoring of RAG1 at the recombination signal nonamer and RAG2-dependent surveillance of adjoining DNA for suitable spacer and heptamer sequences.

extending 2 kb on either side of miRNA166 were identified from GenBank by BLAST analyses. Pairwise comparisons between MIR166 genes were made using Pustell DNA matrix analysis (MacVector 6.5.3). GenBank accession numbers are: rld1, AY501430; mir166a, AY501431; mir166b, AY501432; mir166c, AY501433; mir166d, AY501434. In situ hybridizationTissue sections prepared from shoot apices of two-week-old mutant and wild-type seedlings were pretreated and hybridized as described 27 . Digoxigenin-labelled probes were prepared by in vitro transcription (Stratagene) according to the manufacturer's protocol. An rld1-specific probe encompassing nucleotides 619-1,674 of the coding sequence, and a phb-specific probe encompassing amino acids 462-606 of the Arabidopsis PHB protein were used at a concentration of 0.5 ng ml 21 kb 21 probe complexity. miRNA166 expression was determined using a mir166a-derived probe amplified with primers 6F and 7R. A precursor-specific probe was amplified using primer 7R and a gene-specific primer (CCTCCATCAGATGAGCTCC) downstream of miRNA166.

V(D)J recombination is a form of site-specific DNA rearrangement through which antigen receptor genes are assembled. This process involves the breakage and reunion of DNA mediated by two lymphoid cell-specific proteins, recombination activating genes RAG-1 and RAG-2, and ubiquitously expressed architectural DNA-binding proteins and DNA-repair factors. Here I review the progress toward understanding the composition, assembly, organization, and activity of the protein-DNA complexes that support the initiation of V(D)J recombination, as well as the molecular basis for the sequence-specific recognition of recombination signal sequences (RSSs) that are the targets of the RAG proteins. Parallels are drawn between V(D)J recombination and Tn5/Tn10 transposition with respect to the reactions, the proteins, and the protein-DNA complexes involved in these processes. I also consider the relative roles of the different sequence elements within the RSS in recognition, cleavage, and post-cleavage events. Finally, I discuss alternative DNA transactions mediated by the V(D)J recombinase, the protein-DNA complexes that support them, and factors and forces that control them.

V(D)J recombination occurs at recombination signal sequences (RSSs) containing conserved heptamer and nonamer elements. RAG-1 and RAG-2 initiate recombination by cleaving DNA between heptamers and antigen receptor coding segments. RAG-1 alone contacts the nonamer but interacts weakly, if at all, with the heptamer. RAG-2 by itself has no DNA-binding activity but promotes heptamer occupancy in the presence of RAG-1; how RAG-2 collaborates with RAG-1 has been poorly understood. Here we examine the composition of RAG-RSS complexes and the relative contributions of RAG-1 and RAG-2 to heptamer binding. RAG-1 exists as a dimer in complexes with an isolated RSS bearing a 12-bp spacer, regardless of whether RAG-2 is present; only a single subunit of RAG-1, however, participates in nonamer binding. In contrast, multimeric RAG-2 is not detectable by electrophoretic mobility shift assays in complexes containing both RAG proteins. DNA-protein photo-cross-linking demonstrates that heptamer contacts, while enhanced by RAG-2, are mediated primarily by RAG-1. RAG-2 cross-linking, while less efficient than that of RAG-1, is detectable near the heptamer-coding junction. These observations provide evidence that RAG-2 alters the conformation or orientation of RAG-1, thereby stabilizing interactions of RAG-1 with the heptamer, and suggest that both proteins interact with the RSS near the site of cleavage.Immunoglobulin and T-cell receptor genes are assembled by rearrangement of antigen receptor gene segments during lymphocyte development. This process, termed V(D)J recombination, is mediated by recombination signal sequences (RSSs) composed of conserved heptamer and nonamer elements, separated by spacers of 12 or 23 bp (12-RSSs and 23-RSSs, respectively); recombination normally occurs between gene segments whose RSSs bear spacers of different length (the 12/23 rule). DNA rearrangement is initiated by the recombination activating proteins RAG-1 and RAG-2 (21, 30), which act in concert to introduce a double-strand break (DSB) at the junction between the RSS and the adjacent coding DNA (14,38). This reaction proceeds in two steps: in the first, a nick is introduced at the 5Ј end of the heptamer element flanking the coding DNA; in the second, the resulting 3Ј hydroxyl on the coding end attacks a phosphodiester on the opposite strand (14). As a result, two DNA ends are produced: a signal end, terminating in a blunt, 5Ј-phosphorylated DSB, and a coding end, terminating in a DNA hairpin (14,23,25,31,38,39).Several lines of evidence indicate that V(D)J recombination is a specialized form of DNA transposition (24). These include (i) chemical similarity between RAG-mediated DSB formation and Mu transposition (39), (ii) an analogy between hybrid joint formation and the retroviral disintegration reaction (16), (iii) the ability of the RAG proteins to catalyze integration of signal ends into nonhomologous DNA (1, 9), and (iv) the involvement of hairpin intermediates in the transposition of Tn10 (10). A deeper appreciation of the similarity betwee...