Intermolecular V(D)J Recombination

Abstract

V(D)J recombination plays a prominent role in the generation of the antigen receptor repertoires of B and T lymphocytes. It is also likely to be involved in the formation of chromosomal translocations, some of which may result from interchromosomal recombination. We have investigated the potential of the V(D)J recombination machinery to perform intermolecular recombination between two plasmids, either unlinked or linked by catenation. In either case, recombination occurs in trans to yield signal and coding joints, and the results do not support the existence of a mechanistic block to the formation of coding joints in trans. Instead, we observe that linearization of the substrate, which does not alter the cis or trans status of the recombination signals, causes a specific and dramatic reduction in coding joint formation. This unexpected result leads us to propose a "release and recapture" model for V(D)J recombination in which coding ends are frequently released from the postcleavage complex and the efficiency of coding joint formation is influenced by the efficiency with which such ends are recaptured by the complex. This implies the existence of mechanisms, operative during recombination of chromosomal substrates, that act to prevent coding end release or to facilitate coding end recapture.V(D)J recombination is the process of assembly of T-cell receptor and immunoglobulin genes from V, J, and sometimes D gene segments (1). Lymphoid-specific proteins RAG1 (recombination activating gene 1) and RAG2 (2, 3) and multiple ubiquitously expressed protein factors are essential for V(D)J recombination. RAG1 and RAG2 bind to and cleave at specific recombination signal sequences (RSSs) 1 generating blunt signal ends and covalently sealed, hairpin coding ends (4 -7). RSSs consist of conserved heptamer and nonamer sequences separated by a nonconserved spacer 12 or 23 base pairs long (12-and 23-RSS). V(D)J recombination occurs efficiently in vivo only between RSSs with different spacer lengths, a restriction known as the 12/23 rule (8).Recent biochemical studies indicate that V(D)J recombination proceeds through a series of protein-DNA complexes. Prior to cleavage, the RAG1 and RAG2 proteins form stable complexes with individual and synapsed pairs of RSSs (9 -17). After cleavage, the RAG proteins remain tightly bound to a synapsed pair of signal ends (15, 18) and also appear to interact with the two hairpin coding ends, albeit with lower affinity (15). Therefore, the immediate product of cleavage is thought to be a "cleaved signal complex" containing the four free ends (15).Usually, RSSs involved in V(D)J recombination are located on the same chromosome, i.e. in cis. However, V(D)J recombination has been implicated in the formation of chromosomal translocations leading to lymphoid malignancies (19 -23). In certain cases, such translocations could be the result of V(D)J recombination occurring in trans, using RSSs on different chromosomes. There have been attempts to address this issue. The first such study attempted, and fai...

In Vivo Reinsertion of Excised Episomes by the V(D)J Recombinase: A Potential Threat to Genomic Stability

“…In the case of recombination between two standard coding-segment RSSs, the frequency of trans -V(D)J recombination has been previously shown to be reduced compared to cis -V(D)J recombination events [20,21]. In the present case of recombination between an ESJ and its RSS target, the presence of the second RSS in the SJ could structurally impede—or on the contrary stimulate—RAG fixation and/or activity on the reactive one, and modify the overall recombination efficiency.…”

Activation of V(D)J Recombination Induces the Formation of Interlocus Joints and Hybrid Joints in scid Pre-B-Cell Lines

“…On the other hand, by increasing the local concentrations of two separate recombination substrates, Tevelev and Schatz have observed a higher level of intermolecular joining products (50). The formation of these products appears to follow the 12/23 rule, i.e., requiring synaptic pairing and cleavage between the 12-RSS on one molecule and the 23-RSS on the other molecule (50).…”

A non-B-DNA structure at the Bcl-2 major breakpoint region is cleaved by the RAG complex

“…However, when we included a pair of 12-and 23-signals, Mbr recombination specific to the 23-signal was readily detectable. The simplest explanation for this is that one or both of the coding ends are released during a 12/23 paired V(D)J recombination event 13 and simultaneously a break at the Bcl-2 Mbr is generated by the RAGs. Subsequently, the break at the Mbr is joined to a coding end from the failed V(D)J recombination reaction in ways that are diagrammatically indistinguishable from the t(14;18) translocation ( Supplementary Fig.…”

“…We addressed this issue in the budding yeast S. cerevisiae, whose kinetochores possess only a single microtubule-binding site 12 and whose centromeres (the DNA underlying the kinetochore) have been pinpointed to sequences no longer than 120 base pairs (bp) 13 . Their activity can therefore be turned off by transcription from an adjacent promoter 14 .…”