Figure 5

The RAG and HMGB1 proteins exhibit similar DNA binding activity in Mg²⁺ and Mn²⁺, but Mn²⁺ fails to support stable RAG synaptic complex formation. (A-C). Radiolabeled 23-RSS substrate was incubated with purified cMR1/cMR2 in binding reactions containing of Mg²⁺ or Mn²⁺in the absence or presence of a fixed amount of full-length HMGB1 (300 ng) or increasing amounts (0.1, 10, 100 or 300 ng) of HMG-box A or box B (A), mtA or mtB HMGB1 (B), or BA Tailless or AB Tailless (C). Bound and free DNA were separated by EMSA and protein-DNA complexes visualized from dried gels using a Storm 860 phosphorimager. (D) Purified cMR1/cMR2 was incubated with a radiolabeled 23-RSS substrate in binding reactions containing Ca²⁺, Mg²⁺, or Mn²⁺ with or without added HMGB1 (300 ng) and cold 12-RSS partner as indicated. Protein-DNA complexes were visualized as in (A-C). Assembly of the higher-order paired complex (PC) requires the presence of both HMGB1 and partner RSS, and is formed in the presence of Ca²⁺ and Mg²⁺, but not Mn²⁺. (E-F). Radiolabeled 23-RSS substrate was incubated with purified cMR1/cMR2 in binding reactions containing Mg²⁺ or Mn²⁺ with or without 12-RSS partner and either a fixed amount of HMGB1 or increasing amounts of HMG-boxA (E) or mtA HMGB1 (F). Protein-DNA complexes were visualized as in (A-C).