V(D)J recombination: signal and coding joint resolution are uncoupled and depend on parallel synapsis of the sites.

Abstract

V(D)J recombination in lymphoid cells is a site-specific process in which the activity of the recombinase enzyme is targeted to signal sequences flanking the coding elements of antigen receptor genes. The order of the steps in this reaction and their mechanistic interdependence are important to the understanding of how the reaction fails and thereby contributes to genomic instability in lymphoid cells. The products of the normal reaction are recombinant joints linking the coding sequences of the receptor genes and, reciprocally, the signal ends. Extrachromosomal substrate molecules were modified to inhibit the physical synapsis of the recombination signals. In this way, it has been possible to assess how inhibiting the formation of one joint affects the resolution efficiency of the other. Our results indicate that signal joint and coding joint formation are resolved independently in that they can be uncoupled from each other. We also find that signal synapsis is critical for the generation of recombinant products, which greatly restricts the degree of potential single-site cutting that might otherwise occur in the genome. Finally, inversion substrates manifest synaptic inhibition at much longer distances than do deletion substrates, suggesting that a parallel rather than an antiparallel alignment of the signals is required during synapsis. These observations are important for understanding the interaction of V(D)J signals with the recombinase. Moreover, the role of signal synapsis in regulating recombinase activity has significant implications for genome stability regarding the frequency of recombinase-mediated chromosomal translocations.The exons encoding the immunoglobulin and T-cell receptor variable region domains are assembled during lymphocyte ontogeny by V(D)J recombination (reviewed in references 24 and 34). This site-specific reaction is directed by a pair of joining signals recognized by components of the recombination activity. The consensus sequence of each signal consists of a palindromic heptamer and an AST-rich nonamer. These two elements are separated by a 12-base spacer at one signal and a 23-base spacer at the other. The recombination crossover point for each signal is at the end of the heptamer on the side distal from the nonamer.During recombination, coding ends and signal ends are generated by recombinase-mediated cleavage at the crossover sites. The two coding ends are joined to form what is termed a coding joint, and the two signal ends are joined to form a signal joint. Modification of the coding ends occurs by terminal deoxynucleotide transferase-associated nucleotide addition (7,19,29), by P-element addition (18,29), and by nucleolytic chewback of the exposed ends prior to joint resolution (reviewed in references 21 and 24). These modifications are important to the diversification of the antigenbinding repertoire.Elucidating the order of the steps in this reaction and their mechanistic interdependence is important to our understanding of how the reaction fails and thereby contributes to...

Single-molecule analysis of RAG-mediated V(D)J DNA cleavage

“…Bead release activity can be ablated by decreasing inter-RSS separation from 137 to 73 bp (Fig. S7 D and E), which would be predicted to interfere with RSS synapsis (28) and has been shown to reduce cleavage in biochemical assays (29). Notably, the block to bead release imposed by the 73-bp intersignal distance is overcome when Mn 2+ is used in place of Mg 2+ in the reaction buffer (Fig.…”

The 12/23 rule is enforced at the cleavage step of V(D)J recombination in vivo

“…Several lines of evidence provide additional support for a synapsis model. Previous experiments demonstrated that shortening the distance between the RSS to less than 100 bp, which would physically hinder synapsis, also inhibits product formation (Lewis & Hesse 1991;Sheehan & Lieber 1993;Akamatsu et al 1994). Similar results have recently been obtained in a cell-free system (Eastman et al 1996).…”

“…Analysis of artificial recombination substrates in cultured cells has shown that recombination is severely inhibited when the distance between the RSS is decreased to less than 100 nucleotides, a distance which should inhibit synapsis (Lewis & Hesse 1991;Sheehan & Lieber 1993;Akamatsu et al 1994). Further evidence supporting a requirement for synapsis is provided by certain V(D)J-related events which, in principle, would require cleavage at only a single RSS.…”

DNA Double-Strand Break Repair

“…Third, the efficiency of signal joint completion appears to be less than 100% (5,6). And fourth, coding joints appear to form under conditions where signal joints are unlikely to form efficiently (23,33 There is some feature of the sonicated salmon sperm DNA that may affect V(D)J recombination or the transfection procedure (Tables 1 and 2). At this point, it is difficult to know how the carrier DNA is adversely affecting precision at the signal joint.…”

“…Four of these are representatives of each of the four double-strand break repair complementation groups that have been identified in Chinese hamster cells (8,14,38,40). The remaining two lines are X ray sensitive but not double-strand break repair deficient (15,39 (11,23,33). These substrates all undergo V(D)J recombination in hamster fibroblasts.…”