Modeling of the RAG Reaction Mechanism

Abstract

SUMMARY The RAG complex in vertebrate V(D)J recombination must coordinate its catalytic steps with its binding to its two distant recombination sites in a manner that is still unclear. Here we test the ability of the plausible reaction schemes to fit observed time courses for RAG nicking and DNA hairpin formation inV(D)J recombination. The reaction schemes with the best fitting capability (a) strongly favor a RAG tetrameric complex ([RAG12:RAG22]) over a RAG octameric complex ([RAG14:RAG24]);(b) indicate that once a RAG complex brings two RSS sites into synapsis, the synaptic complex rarely disassembles (nearly irreversible); (c) predict that binding of both RSS sites (synapsis) occurs before catalysis (nicking); and (d) show that RAG binding properties permit strong distinction between RSS sites within active chromatin versus non-specific DNA or RSS sites within inactive chromatin, a point with evolutionary implications. The results provide insights for synapsis by nuclear proteins generally and testable predictions.

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

“…S4B), which is consistent with previous studies (6,(24)(25)(26). We also find the K d values for the 12RSS and the 23RSS in the presence of RAG1/2c and HMGB1 are within error of K d values previously reported that were derived from the turnover number (k cat ) (27).…”

“…This observation is consistent with the HMGB1 dependence of the large bends detected in 12RSS-RAG-HMGB1 or 23RSS-RAG-HMGB1 complexes, although those studies used short oligonucleotide substrates (25,26). We also used this reduction in DNA tether length to determine the K d for the 12RSS and the 23RSS in the presence of RAG1/2c and HMGB1 and found that HMGB1 decreased both similar, consistent with previous observations (6,27). Our singlemolecule assay to study HMGB1-dependent processes could be extended to study other proteins other than RAG1/2c, which has binding to DNA that is also facilitated by HMGB1 (for example, the tumor suppressor protein p53) (32).…”

“…The paired complex was relatively short-lived, which is at odds with kinetic models derived from cleavage experiments with oligonucleotide RSS substrates that predict slow hairpin formation with a long-lived paired complex (27). Furthermore, the paired complex did not always result in a cleaved DNA product; only roughly 40% of paired complexes terminated in a DNA cleavage event.…”

Recruitment of RAG1 and RAG2 to Chromatinized DNA during V(D)J Recombination

“…We also note that our recent ChIP-seq analysis using mouse thymocytes revealed that RAG1 binding was reduced in intensity and less well focused at regions of high H3K4me3 levels in the absence of RAG2 than in its presence (38). These results (which represent a steady-state analysis) are more in keeping with the models derived from in vitro kinetic studies (44). This finding and our previous finding that RAG1 binding at the Igh recombination center was strongly dependent on RAG2 (25) suggest that there are circumstances under which RAG2 can enhance RAG1 binding.…”

“…3D). Our failure to detect cooperation between the RAG proteins in initial binding at J, while largely consistent with data from our previous ChIP-qPCR study (25), was notable in light of a recent in vitro kinetic analysis of RAG-mediated DNA cleavage, which predicted cooperation between RAG1 and RAG2 by virtue of their ability to recognize the RSS (RAG1) and H3K4me3 (RAG2) (44). This discrepancy might relate to differences in interactions with naked DNA in vitro and with chromatin in vivo as well as the need for RAG1 and RAG2 to associate with one another at their relatively low concentrations in vivo (45).…”