Identification of Basic Residues in RAG2 Critical for DNA Binding by the RAG1-RAG2 Complex  Sebastian D Fugmann, David G Schatz  Molecular Cell  Volume 8, Issue 4, Pages 899-910 (October 2001) DOI: 10.1016/S1097-2765(01)00352-5

Figure 1 Coupled Cleavage Activities of RAG2 Mutants Comparison of the cleavage activity of wild-type RAG2 to RAG2 proteins mutate (A) in individual serine, threonine, and tyrosine residues or (B) in individual basic residues. The 12/23-coupled cleavage reactions were performed in 1 mM Mg2+ using a body-labeled 531 bp DNA substrate containing a 12-RSS and a 23-RSS (white and black triangles, respectively). The cleavage products were displayed on 4% native polyacrylamide gels. The structures of the substrate and the reaction products are indicated at the right. The slow migrating band visible in some lanes is a circular intramolecular transposition product (Agrawal et al., 1998). The * indicates a product resulting from aberrant cleavage. The coding flanks and RSSs in this substrate were 5′-GTCGA-h-GTACAGACTGGA-n-3′ (12RSS) and 5′-GGATC-h-ATTCATATCACTGCGCCCCCGTT-n-3′ (23RSS) respectively (h, a consensus heptamer; n, a consensus nonamer) Molecular Cell 2001 8, 899-910DOI: (10.1016/S1097-2765(01)00352-5)

Figure 2 RSS Binding Activity of RAG2 Mutants In Vitro Analyses of the abilities of RAG2 proteins mutated (A) in individual serine, threonine, and tyrosine residues or (B) in individual basic residues to participate in 12-SC formation. Binding assays were performed using GST-RAG2 and MBP-RAG1 proteins in 5 mM Ca2+ with a 5′-end-labeled double-stranded 12-RSS oligonucleotide substrate. The complexes were resolved on native 4% polyacrylamide gels. The * indicates a RAG2 (but not RAG1) containing shifted complex (S.D.F and D.G.S, unpublished data), that appears only in certain preparations of GST-RAG2. The coding flank and RSS in the binding substrate were 5′-TCTTA-h-ATACAGACCTTA-n-3′ Molecular Cell 2001 8, 899-910DOI: (10.1016/S1097-2765(01)00352-5)

Figure 3 Catalytic Activity of RAG2 Mutants for Nicking and Hairpin Formation on Isolated RSS Substrates (A) Nicking. Cleavage reactions were performed in 0.5 mM Mg2+ with a 5′-end-labeled double-stranded 12-RSS oligonucleotide substrate. (B) Hairpin formation. Cleavage reactions were performed in 1 mM Mn2+ with a prenicked version of the 12-RSS substrate in which a nick exists in the top strand between the heptamer and coding flank (lane 1). Reactions contained individually expressed GST-RAG1 and GST-RAG2. The reaction products were displayed on denaturing 8% polyacrylamide gels. The structure of the products is indicated on the right side. The cleavage experiments were repeated using bacterially expressed MBP-RAG1 instead of GST-RAG1, and similar results were obtained (but the overall level of cleavage was reduced) Molecular Cell 2001 8, 899-910DOI: (10.1016/S1097-2765(01)00352-5)

Figure 4 RAG1-RAG2 Interaction in Crude Cell Extracts (A) Western blot analysis of RAG1 and RAG2 expression levels in crude lysates from 293T cells transiently transfected with the indicated combinations of strep-RAG1 and GST-RAG2. (B) Glutathione sepharose beads were incubated with the crude lysates and washed extensively. The protein complexes that remained bound to the resin were analyzed by Western blot analysis. RAG1 and RAG2 proteins were detected using polyclonal anti-RAG1 and anti-RAG2 antisera. The arrows indicate the position of RAG1 and RAG2, and the * indicates the position of the GST-RAG2 (murine amino acids 384–527) fusion protein Molecular Cell 2001 8, 899-910DOI: (10.1016/S1097-2765(01)00352-5)

Figure 5 V(D)J Recombination and RSS Binding of RAG2 Mutants In Vivo (A) Signal and coding joint formation. The ability of wild-type and mutant RAG2 to catalyze signal and coding joint formation in vivo was determined in standard transient V(D)J recombination assays (Hesse et al., 1987) using the signal joint substrate pSF200 or the coding joint substrate pSF290. Signal and coding joint values are displayed on the left and right vertical axes, respectively. The RSSs in the recombination substrates were 5′-ACCTG-h-CTACAGACTGGA-ACAAAAACA-3′ (12-RSS) and 5′-ACCTG-h-GTAGTACTCCACTGTCTGGCTGT-n-3′ (23-RSS). (B) One-hybrid DNA binding assay. The ability of RAG1 and RAG2 to bind the 12-RSS was measured using a mammalian one-hybrid system, described previously (Difilippantonio et al., 1996). The firefly and renilla luciferase activities of crude lysates of cells transfected with RAG1, RAG2-VP16 fusion proteins, the p(12)8 reporter plasmid, and pRL-CMV (expressing renilla luciferase) were determined. The firefly luciferase values were normalized by dividing them by the Renilla luciferase values, which corrects for variations in transfection efficiency (see Experimental Procedures). The background luciferase activity observed in cells without RAG proteins was arbitrarily set to 1.0, and all other values are expressed relative to this. For both (A) and (B), values represent the mean of results from two independent transfections, and error bars indicate the standard error of the mean. The RSS used in the binding substrates was 5′-CTCGA-h-GTACAGACTGGA-n-3′ Molecular Cell 2001 8, 899-910DOI: (10.1016/S1097-2765(01)00352-5)

Figure 6 Structural Model for RAG2-DNA Interactions (A) Ribbon diagram (side and top views) of the predicted structure for RAG2 (Corneo et al., 2000). β strands are shown in blue, and the five basic residues important for DNA binding, K119, H140, R159, R229, and K283, are displayed as red ball and stick representations. While K119, R159, R229, and K283 are located on the surface of the globular structure, H140 is predicted to be buried in the hydrophobic core and is, thus, less likely to be involved in direct RAG2 DNA contacts. (B) Surface potential of the predicted RAG2 structure (side and top views, identical to those in [A]). The electrostatic surface potential of the predicted RAG2 structure was calculated and displayed using the SwissPDBviewer (Guex and Peitsch, 1997). Positively charged (basic) areas are indicated in blue, negatively charged (acidic) in red. K119, R159, and K283 contribute to the positively charged surface patch, and the positions of K119, R159, and K283 are indicated by orange circles in both parts of the figure Molecular Cell 2001 8, 899-910DOI: (10.1016/S1097-2765(01)00352-5)