Figure 8

RAG-mediated bps6197 cleavage in cells is not detectable. (A) Diagrams showing the plasmid V(D)J recombination substrate pJH299 (12/23) or its derivatives in which the 23-RSS is replaced by bps6197 in the same orientation (12/6197SO) or reverse orientation (12/6197RO). SEBs produced by RAG-mediated cleavage were detected by LM-PCR as described in Figure 1. (B) (top) The plasmid substrates in (A), linearized with Aat II, were incubated with D600A or WT cMR1/cMR2 in an in vitro cleavage reaction lacking or containing HMGB1, and RSS or bps6197 SEBs were detected by LM-PCR as in Figure 1B. (Bottom) Plasmid V(D)J recombination substrates in (A) were cotransfected with WT or D600A cMR1 and WT cMR2 expression constructs into 293 cells. Plasmid DNA recovered 72 h post-transfection was analyzed for the presence of RSS or bps6197 SEBs by LM-PCR using the strategy in (A). To verify the presence of template DNA, a fragment of the chloramphenicol acetyl transferase (CAT) gene in the pJH299 backbone was amplified by PCR as a control.