Figure 1

Proteins and DNA substrates used in this study. (A) Diagram of proteins used in this study. Maltose binding protein (MBP) tagged forms of truncated RAG1 (384–1040) and RAG2 (1–387) (cMR1 and cMR2, respectively) were coexpressed in 293 cells and purified by amylose affinity chromatography. Full-length, truncated, and mutant forms of recombinant HMGB1 are illustrated below a diagram indicating the residues encompassing the amino-terminal polyhistidine tag (His₆), the HMG box domains (rectangles), the basic linker (heavy line) and the acidic tail (oval). Mutant forms of full-length HMGB1 contain ten consecutive alanine substitutions (A₁₀) in box A (mtA HMGB1, residues 18–27) or box B (mtB HMBG1, residues 102–111). Tailless forms of HMGB1 contain box A and box B in their wild-type configuration (AB Tailless) or are in reverse order (BA Tailless). (B) Diagram of the 23-RSS substrate used in this study. The substrate is radiolabeled on the top strand at the 5'-end (³²P) and the heptamer and nonamer motifs are shaded. The lengths of the flanking and spacer regions are also indicated. RAG-mediated cleavage proceeds via a two-step mechanism involving top strand nicking at the 5'-end of the heptamer, followed by direct transesterification to liberate a blunt, 5'phosphorylated signal end, and a coding end terminating in a DNA hairpin structure [3].