Correlation between recombinase activating gene 1 ubiquitin ligase activity and V(D)J recombination

Abstract

The really interesting new gene (RING) finger ubiquitin ligase domain of the recombinase activating gene 1 (RAG1) V(D)J recombinase protein adopts a standard cross-brace architecture but co-ordinates three zinc ions as opposed to the canonical two. We demonstrated previously that disruption of the conserved zinc co-ordination sites resulted in loss of structural integrity and ubiquitin ligase (E3) activity and interfered with the ability of full-length RAG1 to support recombination. Here we present evidence that amino acids surrounding the third, non-canonical site also contribute to functional interaction with the ubiquitin conjugating (E2) enzyme CDC34, while certain residues on the RING domain's surface important for interaction between other E2-E3 pairs are less critical to the functional RAG1-CDC34 interaction in this assay. Partial reduction of ubiquitin ligase activity was significantly correlated with reduction in the ability of RAG1 to support recombination of extra-chromosomal substrates (r = 0.805, P = 0.009). While poly-ubiquitin chains could be generated, RAG1 did not promote rapid chain extension following mono-ubiquitylation of substrate, regardless of the E2 enzyme used. No single ubiquitin lysine mutant disrupted the ability of CDC34 to form ubiquitin chains on RAG1, and mass spectrometric analysis of the poly-ubiquitylated products indicated ubiquitin chain linkages through lysines 48 and 11. These data suggest that RAG1 promotes a mono-ubiquitylation reaction that is required for optimal levels of V(D)J recombination.

Figure 1

Diagram of the recombinase activating gene 1 (RAG1) and cbl zinc-binding domains. (a) Zinc-binding residues of the RAG1 really interesting new gene (RING) domain are shown, including the co-ordination of the third zinc ion by the non-canonical binding residues C266, H270 and H295. The first zinc site is co-ordinated by residues C290, C293, C310 and C313. The second zinc site is co-ordinated by residues C305, H307, C325 and C328. (b) The cbl diagram represents a classic RING domain with the first zinc co-ordinated by C381, C384, C401 and C404. The second zinc is co-ordinated by residues C395, H398, C416 and C419.

Figure 2

In silico alignment of really interesting new gene (RING) structures. Backbone traces of the recombinase activating gene 1 (RAG1) RING structure with the cbl RING structure determined as a part of the cbl–UbcH7 complex are shown., Red, UbcH7; green, cbl; yellow, RAG1; magenta spheres, cbl zinc ions; grey spheres, RAG1 zinc ions. Protein Data Bank (PDB) ID for the cbl–ubcH7 co-structure is 1fbv, PDB ID for RAG1 RING is 1rmd.

Figure 3

Ubiquitin ligase activity of recombinase activating gene 1 (RAG1) mutants. (a) Sample auto-ubiquitylation assay performed as described in the Materials and methods using RAG1[218–389] wild type (WT) and CDC34 as indicated. Products were analysed by western blot with anti-Xpress antibody followed by anti-mouse-horseradish peroxidase (HRP) antibody. The positions of unmodified (R1), mono- and poly-ubiquitylated RAG1 are indicated. (b) Karyopherin alpha 1 (KPNA1) ubiquitylation assays were performed as described in the Materials and methods using the E2 enzyme UbcH2 or UbcH5a and RAG1[218–389] WT. The percentage of KPNA1 converted to mono- (Ubi₁), di- (Ubi₂), tri- (Ubi₃) and tetra-ubiquitylated (Ubi₄) species was determined by western blot for each sample. The average and standard deviation for three independent trials are shown. (c) Ubiquitin ligase assays were performed as described in (a) using the WT or various mutants of RAG1[218–389]. The percentage of RAG1 converted to mono- (Ubi₁), di- (Ubi₂), tri- (Ubi₃) and tetra-ubiquitylated (Ubi₄) species was determined by western blot for each sample. The average and standard deviation for at least three independent trials are shown; the significance of the decrease in auto-ubiquitylation relative to WT was determined by Student’s t-test (*P < 0·05; **P < 0·01). I/C/S, triple mutant I292A/C317A/S327F. (d) Partial proteolysis was performed with trypsin as described in the Materials and methods. Products were separated by sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS-PAGE) on an 18% gel and analysed by western Blot as described previously. Open arrows, undigested RAG1[218–389]; filled arrows, cleavage product spanning amino acids 254–377. Please note that extraneous lanes have been excised between the I/C/S and S327F samples and between H270A and N265A.

Figure 4

Activity of various ubiquitin lysine mutants. (a) Auto-ubiquitylation assays were performed as described in the Materials and methods, with protein kinase tagged ubiquitin (PK-Ubi) or methylated ubiquitin [(CH₃)-Ubi] as indicated. Products were analysed by western blot with anti-Xpress antibody followed by anti-mouse-horseradish peroxidase (HRP) antibody. (b) The recombinase activating gene 1 (RAG1)[218–389] ubiquitylation assay was performed with PK-Ubi; products were separated on a 4–12% NuPAGE gel in 3-(N-Morpholino)propanesulphonic acid, 4-Morpholinepropanesulphonic acid (MOPS) buffer (Invitrogen) alongside Mark 12 molecular weight (MW) standards (Invitrogen), stained with SimpleBlue and cut into slices as indicated (J1–J15). (c) Assays were performed as in (a) with RAG1[218–389] and various ubiquitin lysine mutants as indicated. R1₂, RAG1 dimer.

Figure 5

Substrate-dependent and -independent ubiquitylation. (a) Ubiquitin ligase assays (16 hr) were performed as described in the Materials and methods using recombinase activating gene 1 (RAG1)[218–389], CDC34 and RAG1[264–389] as indicated. Products were analysed by western blot with anti-ubiquitin conjugate antibody followed by anti-rabbit-horseradish peroxidase (HRP) antibody. (b) Staging scheme for reactions shown in (a) lanes 2 and 3. (c) CDC34 charging assays were performed as described in the Materials and methods in the presence of RAG1[264–389] or RAG1 storage buffer as indicated. The percentage CDC34 charged was determined by western blot. The average and standard deviation for three independent trials are shown. R1, RAG1.

Figure 6

Recombination activity of recombinase activating gene 1 (RAG1) mutants. Signal joint (SJ) (a) and coding joint (CJ) (c) assays were performed as described in the Materials and methods, with RAG1 wild type (WT), N265A, H270A, I292A, E294A, S327F or I292A/C317A/S327F (I/C/S) as indicated. Recombination was assessed by polymerase chain reaction (PCR); the average and standard deviation were calculated from three independent trials. The significance of recombination impairment relative to WT was assessed by Student’s t-test (*P < 0·05; ***P ≤ 0·001). (b) Correlation between deficits in recombination and E3 activity was assessed by regression analysis. The data points indicated by small arrows are from previously published work; remaining data points are from this paper.