Figure 2

Individual HMG-box domains stimulate RAG-mediated hairpin formation in Mn²⁺, but not Mg²⁺, in the absence of synapsis, but addition of partner RSS bypasses this impairment in Mg²⁺. (A-B) Radiolabeled 23-RSS substrate was incubated with purified cMR1/cMR2 in an in vitro cleavage reaction containing of Mg²⁺ (A) or Mn²⁺ (B) with or without added 12-RSS in the absence or presence of increasing amounts (0.1, 10, 100 or 300 ng) of full-length HMGB1, HMG-box A, or HMG-box B at 37°C for 1 hr (Mg²⁺) or 10 min (Mn²⁺) in the combinations indicated above the gel. Reaction products were fractionated on a sequencing gel and visualized using a Storm 860 phosphorimager (left panels). The percentage of hairpin products (% Hairpin) were quantified for each reaction from at least three different gels with the average values (and standard deviations) presented in bar graph format (lower right panels). The percentage of nicks sited at the 5'-end of the heptamer (% Nick), and aberrantly introduced at position 27 (% Abnick; equivalent to nicking the substrate as a 12-RSS) is quantified from the gel shown and presented in bar graph format (open and filled bars, respectively; upper right panels). The results are representative of data obtained from at least three independent experiments.