Figure 2

Efficient RAG-mediated cleavage of bps6197 requires HMGB1 and is stimulated by synapsis with a 12-RSS partner. (A) DNA fragments radiolabelled at the 5' end of the top strand (asterisk) were generated by PCR using pGG49 (bps6197/12/23) or its derivatives as templates (see diagrams; designations are indicated at right). The sizes of the intact bps6197/12/23 substrate and reaction products predicted from RAG-mediated cleavage or restriction enzyme digestion of this substrate are shown. (B) The indicated DNA fragments described in (A) were incubated in an in vitro cleavage reaction with HMGB1 and WT or D600A cMR1/cMR2 and purified reaction products were analyzed on a vertical agarose gel in parallel with a radiolabeled 100 bp ladder (M). Two major cleavage products (A and B, indicated at right) were isolated and used as markers in subsequent experiments. (C) The DNA fragments described in (A) were subjected to in vitro cleavage by WT or D600A cMR1/cMR2 in the absence or presence of HMGB1 as indicated. Reaction products were analyzed on a 40% formamide sequencing gel in parallel with markers derived from restriction enzyme digestion of bsp6197/12/23 or isolated after RAG-mediated cleavage as described in (B). Expected fragment sizes and compositions are indicated at left and right, respectively. The abundance of the major reaction products are quantified below the gel. Comparable results have been obtained in independent experiments.