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

Abstract

The recombination-activating gene products, RAG1 and RAG2, initiate V(D)J recombination during lymphocyte development by cleaving DNA adjacent to conserved recombination signal sequences (RSSs). The reaction involves DNA binding, synapsis, and cleavage at two RSSs located on the same DNA molecule and results in the assembly of antigen receptor genes. We have developed singlemolecule assays to examine RSS binding by RAG1/2 and their cofactor high-mobility group-box protein 1 (HMGB1) as they proceed through the steps of this reaction. These assays allowed us to observe in real time the individual molecular events of RAG-mediated cleavage. As a result, we are able to measure the binding statistics (dwell times) and binding energies of the initial RAG binding events and characterize synapse formation at the single-molecule level, yielding insights into the distribution of dwell times in the paired complex and the propensity for cleavage on forming the synapse. Interestingly, we find that the synaptic complex has a mean lifetime of roughly 400 s and that its formation is readily reversible, with only ∼40% of observed synapses resulting in cleavage at consensus RSS binding sites. The recombination-activating genes, RAG1 and RAG2, encode proteins that carry out V(D)J recombination by bringing a 12RSS and a 23RSS together into a paired (or synaptic) complex, nicking the RSS sites adjacent to the heptamer, and converting each nick into a double-strand break, leaving a hairpin at the coding end (Fig. 1). The hairpin is created by nucleophilic attack on the opposing strand by the 3′ hydroxyl group at the nick in a transesterification reaction. After RAG1/2 forms hairpins, it recruits the nonhomologous end joining machinery to repair the ends (1, 2). Since their discovery, the full-length RAG1 and RAG2 proteins have proven difficult to isolate and study in vitro (1). However, core domains (referred to as RAG1c and RAG2c) have been identified by removing a large region from the N terminus of RAG1 (which includes an E3 ubiquitin ligase domain) and a large region from the C terminus of RAG2 (which includes a plant homeodomain) (3, 4). These core proteins have been shown to tetramerize to form RAG1/2c, which retains RSS binding, nicking, and hairpin formation activities (5). High-mobility group-box protein 1 (HMGB1) acts as a cofactor and increases RAG1/2c affinity for the 23RSS (6). HMGB1 is required for paired complex formation and efficient conversion of RAG-mediated nicks into hairpins (7-10).Current in vitro assays that capture the paired complex with RAG1/2c generally place a 12RSS and a 23RSS on two different DNA molecules, typically short oligonucleotides. However, in vivo, antigen receptor loci are assembled using RSSs on the same DNA molecule (1, 11). One prior study captured RAG1/2c and HMGB1 bound to DNA with a 12RSS and a 23RSS on the same substrate using standard bulk assays (12). However, many key mechanistic questions remain unresolved concerning how a diverse immune repertoire is generated by the RAG recombinas...

Real-time analysis of RAG complex activity in V(D)J recombination

“…To determine HMGB1's role as a cofactor, we conducted experiments with both 50 nM RAG1/2 and 50 nM HMGB1 in solution. Previous studies have hypothesized roles for HMGB1 in stabilizing the RAG1/2 nucleoprotein complex and increasing RAG1/2 binding affinity for RSS substrates (21,22). We found that FRET distribution for the 12RSS substrate was narrowed in the presence of RAG-HMGB1 complex in comparison with RAG1/2-only conditions (Fig.…”

“…The effect of RAG1/2 complex binding to RSS DNA was previously addressed using biochemical and biophysical techniques (19,20,22,26) and more recently visualized within the PC in structural analysis (see SI Discussion) (14). These studies indicated that binding of HMGB1 and RAG1/2 induced bending in both the 12RSS and 23RSS DNA substrates as well as in each substrate within the PC.…”

“…This specificity, along with the 12/23 rule, has been the subject of several ensemble studies as well as a recent single-molecule study (22). Although these data provided important characteristics of RAG1/2 DNA binding activity, they were mostly derived from inferential assays.…”

Sequence-Dependent Dynamics of Synthetic and Endogenous RSSs in V(D)J Recombination

“…Synthetic RSSs. We chose a 12RSS flanking the immunoglobulin κ variable (IgκV) gene segment, V4-57-1, as the reference sequence due to its use in a previous TPM study on RAG-RSS interactions (20) for our reference sequence. This sequence has also been used in structural studies of RAG-RSS complexes (13,15), allowing us to compare our results with known information on the RAG-RSS structure.…”

“…2C). TPM has been applied to track the dynamic behavior of various protein-DNA systems, including RAG and RSS (16)(17)(18)(19)(20)(21). It is with the temporal resolution provided by TPM that we can track the full progression of individual RAG-RSS interactions from PC formation to cleavage.…”