The RAG-HMG1 Complex Enforces the 12/23 Rule of V(D)J Recombination Specifically at the Double-Hairpin Formation Step

The central feature of specific immunity is its capability to generate an enormous repertoire of antigen receptors by a process called V(D)J recombination. During this process, any one of an array of V (variable) gene segments can be joined to one of many D (diversity) or J (joining) gene segments to generate a new exon encoding the antigen binding domains of immunoglobulins or T-cell receptors. The cis elements mediating the site specificity of the recombination reaction are recombination signal sequences (RSS) flanking the V, D, and J gene segments. They consist of a palindromic heptamer separated from an A+T-rich nonamer by a spacer of either 12 or 23 bp. A single recombination event is directed by two of these joining signals with different spacer lengths, one with a 12-bp spacer (12-signal) and the other with a 23-bp spacer (23-signal). This restriction is referred to as the 12/23 rule (22). The 12/23 rule ensures that the recombining gene segments are joined together correctly. The recombination reaction can be broadly divided into two phases. The first phase is cleavage at the signal sequences. The second phase is the ligation, or repair, of the gene segment termini to create the new exon. The RAG1 and RAG2 proteins (15, 17, 18) have been found to be necessary and sufficient for cleavage of the recombination signals in vitro (17, 24, 26). After binding and synapsis of the two signals, the reaction occurs by a two-step chemical mechanism during which one DNA strand is nicked at the heptamer of each RSS. The 3′ OH group that is generated attacks the phosphodiester bond of the antiparallel strand in a direct transesterification reaction, leaving hairpinned coding ends and blunt signal ends (25). Recently, a DNA-bending protein, HMG1, was found to markedly enhance the cutting reaction (16, 23). The second phase involves factors that participate in general double-strand break repair. These include the Ku70/86, DNA-PK, XRCC4, and DNA ligase IV (5, 17). Several studies have indicated that the 12/23 rule is enforced at one of the steps of the cleavage phase of V(D)J recombination rather than at a later step. Such studies include exogenous substrate analysis (19, 21) and assays with crude extract cleavage of linear DNA (3, 16). One of these studies used a cell line that overexpressed RAG proteins to create active crude extracts for the cleavage step of the V(D)J reaction (3). In a series of experiments, the authors examined the ability of these lysates to form nicked or hairpinned products on linearized plasmid-based substrates. However, because these studies utilized plasmid-based substrates of several hundred base pairs, it is difficult to discern the relative rates of nicking and hairpin formation. Furthermore, these studies were performed in crude lysates, which prevent accurate measurements of the contribution of the RAG proteins specifically. Examination of the role of RAG proteins for establishing the 12/23 rule has been problematic. Specifically, the levels of adherence to the 12/23 rule between RAG proteins overexpressed in crude cell lysates and as purified proteins have been found to differ (16). RAG proteins in crude lysates were shown to have a rate of cutting on 12×23 plasmid substrates at least 25-fold greater than that on non-12×23 substrates (3). Under similar conditions, purified RAG proteins showed only a two to fourfold preference for the 12×23 substrate (16, 26). This important discrepancy has not yet been reconciled. We generated a novel system that allows the examination of concerted cutting at two recombination signal sequences in a much more detailed manner than was possible with previous systems. Under these conditions, we demonstrated that purified RAG proteins and HMG1 have a strict adherence to the 12/23 rule similar to that of RAG proteins in crude lysates. Having demonstrated that this system contains the essential components to reproduce the 12/23 rule, we went on to study the interaction of the RAG proteins with the 12×23 and alternative 12×12 and 23×23 substrates at each step of the cleavage reaction. We found that binding and nicking by the RAG proteins was no different for the 12×23 and the alternative substrates. However, at the nick to hairpin transition, the RAG proteins were unable to efficiently cleave the 12×12 or 23×23 substrates or prenicked 12×12 or 23×23 intermediates. The only substrates successfully cleaved were those with both the 12-signal and the 23-signal, establishing that the 12/23 rule is enforced at the formation of the double hairpin structure.