Figure 3.

RAG proteins purified using the mild procedure exhibit enhanced activity in RSS cleavage assays. Radiolabeled intact 23-RSS substrate was incubated for 1 h at 37°C with cMR1/cMR2 (WT or D600A RAG1), cMR1/FLMR2 or FLMR1/cMR2 purified using standard or mild conditions in cleavage reactions containing Mg²⁺ in the absence or presence of HMGB1 or cold 12-RSS partner as indicated above the gel. Reaction products were fractionated by denaturing gel electrophoresis and analyzed using a phosphorimager running the ImageQuant software. The positions of expected products are indicated at right. The asterisk denotes the location of ³²P on the top strand. The percentage of correctly nicked (%N), aberrantly nicked (%Abnick) and hairpin (%HP) products in each lane is quantified below the gel.