Regulation of RAG1/RAG2-mediated transposition by GTP and the C-terminal region of RAG2

Abstract

The RAG1 and RAG2 proteins perform critical DNA recognition and cleavage functions in V(D)J recombination, and also catalyze efficient DNA transposition in vitro. No transposition in vivo by the RAG proteins has been reported, suggesting regulation of the reaction by as yet unknown mechanisms. Here we report that RAG-mediated transposition is suppressed by physiological concentrations of the guanine nucleotide GTP, and by the full-length RAG2 protein. Both GTP and full-length RAG2 inhibit transposition by blocking the non-covalent 'capture' of target DNA, and both are capable of inhibiting RAG-mediated hybrid joint formation in vitro. We also observe that another intracellular signaling molecule, Ca(2+), stimulates RAG-mediated transposition and is capable of activating transposition even in reactions containing full-length RAG2 and GTP. RAG-mediated transposition has been proposed to contribute to the chromosomal translocations that underlie the development of lymphoid malignancies, and our findings highlight regulatory mechanisms that might prevent such occurrences, and circumstances in which these regulatory mechanisms could be overcome.

Fig. 1. GTP is a specific inhibitor of RAG-mediated transposition. (A) The effect of nucleotides on the cleavage and transposition activities of the RAG1/RAG2 proteins was assessed using a body-labeled cleavage substrate (arrowhead) containing a 12-RSS and a 23-RSS in the presence of 10 mM MgCl₂. Structures of the substrate and reaction products are indicated on the right, with the 12-RSS and 23-RSS depicted as open and filled triangles, respectively, and coding ends as open circles. The concentration of the nucleotides used is indicated above each lane. tnp, intramolecular transposition product. The reactions products were analyzed on a native 4% (acryamide:bis = 29:1) polyacryamide gel and quantitated using a PhosphorImager (Molecular Dynamics). (B) Effect of GTP derivatives (1 mM) on the activities of the RAG proteins was assessed using the same assay as in (A). (C) Structure of GTP with the functional groups critical for GTP inhibition highlighted in shaded circles and the functional groups that are dispensable highlighted in open circles.

Fig. 2. Effect of GTP on the formation of post-cleavage complexes. (A) Schematic diagram of the DNA–RAG1/RAG2 complexes leading to transposition. (B) SEC formation reactions were carried out using a 5′ end-labeled 12-signal end oligonucleotide in a reaction buffer containing 4 mM MgCl₂ in the presence or absence of HMG2 and unlabeled 23-signal end, and various amount of GTP, as indicated above the lanes. Reactions were analyzed on a native 6% (80:1) polyacrylamide gel. (C) TCC formation and intermolecular transposition reactions were carried out in a two-stage fashion. First, the SEC was formed as in (B) except that both the 12-signal end and 23-signal end were unlabeled, and reactions contained nucleotide (1 mM) or 1 mM CaCl₂ as indicated above the lanes. Formation of the TCC and STC were initiated by adding 5′-end-labeled oligonucleotide target DNA. Deproteinized transposition products were visualized by treating the reaction with SDS/EDTA prior to loading on a native 6% (80:1) polyacrylamide gel.

Fig. 3. Identification of a putative GTP-binding domain. (A) Schematic diagram of the murine RAG1 protein depicting three active site residues (D600, D708 and E962), the nonamer-binding domain (NBD), and the putative GTP-binding domain (aa 717–725). A sequence alignment of RAG1, the GTP-binding domain of TGII and the consensus sequence of the P loop is shown, with identical residues shown in gray boxes. (B) Effect of GTP on the activities of the RAG1 mutant G717A was assessed using the assays described in Figure 1A in the presence of various concentrations of GTP as indicated above the lanes, except that the concentration of MgCl₂ was 6 mM. (C) Quantitation of transposition in (B). Transposition activity was calculated as the ratio of the intensity of the transposition product to the sum of the intensities of the signal end fragment and the transposition product.

Fig. 4. Full-length RAG2 exhibits reduced transposition and hybrid joint formation activities compared with core RAG2. (A) Standard in vitro 12/23 coupled cleavage reactions were carried out in the presence of 4 mM MgCl₂ and various concentrations of nucleotides as indicated. The RAG proteins used in the reaction were partially purified from 293T cells co-expressing the GST-core RAG1 fusion protein and either core RAG2 (lanes 2–8) or full-length RAG2 (lanes 9–15). Interaction of these proteins with substrate DNA generates a faint smear at approximately the position of the transposition product (most easily seen in lanes 12 and 13). (B) Schematic diagram depicting the formation of hybrid joints, in which RSSs become linked to the opposite coding end. Half arrows represent PCR primers used for hybrid joint detection. (C) In vitro 12/23 coupled cleavage reactions were carried out in the presence of 1 mM GTP or ATP as indicated for 30 or 60 mins, and reaction products were used as templates for PCR amplification of hybrid joints. Prior to the analyses of hybrid joint formation by PCR, the templates were analyzed for the accumulation of cleavage products and were found to be comparable at all time points (data not shown). Asterisks indicate non-specific PCR products, while arrows indicate hybrid joint products. (D) The SEC formation assay was performed using MBP-core RAG1 together with either GST-core RAG2 (lanes 2–4) or GST-full-length RAG2 (lanes 5–7). (E) The assays for the TCC and STC formation and intermolecular transposition were carried out using the RAG proteins as indicated. The reaction conditions are identical to those described in Figure 2C.

Fig. 5. (A) The effect of CaCl₂ on the activities of the RAG proteins and GTP inhibition of transposition was assayed using standard coupled cleavage assays (4 mM MgCl₂) in the presence or absence of 2 mM GTP and increasing concentrations of CaCl₂ (0.2, 0.5, 1 and 2 mM). (B) Quantitation of the results of (A). (C) The effect of 1 mM CaCl₂ on transposition by core RAG2 and full-length RAG2 in the presence or absence of 1 mM GTP.