Figure 6

Single HMG-box domains and forms of HMGB1 bearing mutations in regions required for its DNA binding and bending activity fail to efficiently stimulate RAG-mediated bps6197 cleavage. (A) Diagrams of full-length, truncated, and mutant forms of recombinant HMGB1 and HMGB2 used in this study. The amino-terminal polyhistidine tag (His₆), the HMG box domains (rectangles), the basic linker (heavy line) and the acidic tail (oval) are indicated schematically. Truncation mutants are shown comprising either a single HMG-box A domain (Box A) or a single HMG-box B domain lacking or containing the basic linker and acidic tail (Box B and B', respectively). Other truncation mutants containing both HMG-box domains lacking the acidic tail only (Basic) or both the basic linker and acidic tail (Tailless) are also shown. A form of Tailless HMGB1 in which the order of the HMG-box domains is reversed was also prepared (Shuffled). Mutant forms of full-length HMGB1 contain ten consecutive alanine substitutions (A₁₀) in box A (mtA, residues 18–27), box B (mtB, residues 102–111), or both (mtAB). (B) The indicated radiolabeled DNA fragments were incubated with WT cMR1/cMR2 in an in vitro cleavage reaction lacking or containing the various forms of HMGB1 or HMGB2 shown in (A). Based on previous studies [26], different amounts of each form of HMGB1 or HMGB2 was added to the cleavage reaction as follows: 600 ng (Box B'), 300 ng (WT, mtA, mtB, and mtAB), 200 ng (Box A and Box B), or 100 ng (Basic, Tailless, Shuffled, and HMGB2). Similar results were obtained in independent experiments.