Abstract
BackgroundFunctional immunoglobulin and T cell receptor genes are produced in developing lymphocytes by V(D)J recombination. The initial site-specific DNA cleavage steps in this process are catalyzed by the V(D)J recombinase, consisting of RAG1 and RAG2, which is directed to appropriate DNA cleavage sites by recognition of the conserved recombination signal sequence (RSS). RAG1 contains both the active site and the RSS binding domains, although RAG2 is also required for DNA cleavage activity. An understanding of the physicochemical properties of the RAG proteins, their association, and their interaction with the RSS is not yet well developed.ResultsHere, we further our investigations into the self-association properties of RAG1 by demonstrating that despite the presence of multiple RAG1 oligomers, only the dimeric form maintains the ability to interact with RAG2 and the RSS. However, facile aggregation of the dimeric form at physiological temperature may render this protein inactive in the absence of RAG2. Upon addition of RAG2 at 37°C, the preferentially stabilized V(D)J recombinase:RSS complex contains a single dimer of RAG1.ConclusionTogether these results confirm that the functional form of RAG1 in V(D)J recombination is in the dimeric state, and that its stability under physiological conditions likely requires complex formation with RAG2. Additionally, in future structural and functional studies of RAG1, it will be important to take into account the temperature-dependent self-association properties of RAG1 described in this study.
Highlights
Functional immunoglobulin and T cell receptor genes are produced in developing lymphocytes by V(D)J recombination
Temperature-dependent self-association properties of MBP-core RAG1 fusion protein (MCR1) Recombinant core RAG1, expressed and purified from bacteria as a fusion protein with maltose binding protein (MBP), was previously shown to be active in in vitro DNA cleavage assays when combined with RAG2 purified from 293T cells [24]
As done previously [21], we focused on the characterization of three fractions of MCR1 that were pooled after the preparative size exclusion chromatography (SEC) gel filtration step (Figure 1)
Summary
Functional immunoglobulin and T cell receptor genes are produced in developing lymphocytes by V(D)J recombination. V(D)J recombination leads to the assembly of genes encoding for the immunoglobulin and T-cell receptors (TCR), and as a result is a fundamental process in the development of lymphocytes [1,2]. Once the RAG proteins have created the double-strand breaks, the appropriate joining of both the coding and signal ends require factors that mediate nonhomologous DNA end joining [4]. The RAG proteins form DNA double strand breaks in two successive catalytic steps [3]. The macromolecular assemblies differ for each of the catalytic steps, with hairpin formation requiring the presence of both the 12 and 23-RSS in the paired complex, whereas nicking may be carried out on a 12-RSS in the single RSS complex [8]. To understand the parameters for both DNA cleavage steps, it is important to characterize both the single RSS and paired complexes. A separate study concluded that at least a trimer of RAG1 was present in both the single RSS and the paired complexes [13]
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