FIG. 4.
Full-length RAG-2 but not full-length RAG-1 suppresses transposition in vitro. A, RAG-mediated transposition of radiolabeled signal ends into a cold target plasmid covalently links the signal ends to the target DNA and linearizes the plasmid. B, wild-type or active site mutant (D600A) cMR1 coexpressed with cMR2 (WT-cMR1/cMR2 or MT-cMR1/cMR2, respectively), cMR1/FLMR2, or FLMR1/cMR2 was incubated with a radiolabeled 23-SE with HMG-1 and cold partner DNA (12-SE), under binding conditions as indicated above the gel, and then subjected to an in-gel transposition assay (see "Experimental Procedures"). An autoradiograph of the DEAE-cellulose paper to which the DNA was transferred is shown here, with the position of the signal end complex (SEC) shown at right. C, reaction products were isolated from the SECs using the autoradiograph in B and analyzed on a native linear 4–20% gradient gel. Linearized 5'-end-labeled pcDNA1 and pre-cleaved substrate (lanes 1 and 2; indicated at right) serve as markers. The percentage of recovered plasmid DNA that is linearized is quantified below the gel (%TP). Similar results were obtained with radiolabeled 12-SE substrates (data not shown).