Figure 7

The V(D)J recombinase:12-RSS complex containing RAG2 and a single RAG1 dimer is preferentially formed at physiological temperature. (A) Left Panel: Radiolabeled 12-RSS was incubated with 0.04 μM MCR1 and 0.14 μM GST-core RAG2 (and 0.34 μM HMGB1 in some samples) at 10°, 25°, or 37°C, as indicated. The supershifted bands in lanes 2–4 that contain RAG2, and in lanes 5–7 that contain both RAG2 and HMGB1, are designated as complexes 1 and 2, respectively, with complex 1 containing 2 RAG1 subunits and complex 2 containing 4 RAG1 subunits. R1 and R2 refer to MCR1 and GST-core RAG2, respectively. H1 refers to HMGB1. Right Panel: Overexposure of autoradiogram to illustrate band positions of R1:RSS complexes (labeled as complex 1 and 2), relative to R1/R2:RSS complexes. Lanes 8 and 9 correspond to lanes 1 and 2 in the left panel. (It should be noted that in lane 8, R1:RSS complexes 1 and 2 correspond to the 2- and 4-subunit MCR1:12-RSS complexes denoted in Figure 4A.) (B) Left Panel: The supershifted RAG1:RAG2 complexes with the 12-RSS are catalytically active as determined by an in-gel cleavage assay (see Methods). The hairpin products in lanes 3 and 6 are more visible upon longer exposures of the gel (not shown). Complexes 1 and 2 refer to the bands as labeled in panel A. For example, lane 4 represents the cleavage products generated from the R1/R2 complex 2 in lane 3 of panel A. Right Panel: Quantification of the percentage of nick and hairpin products (from n = 3 in-gel cleavage assays) relative to the total amount of radioactivity in each lane.