FIG. 3.

Fluorophore-labeled 12- and 23-RSS substrates support RAG-mediated synapsis and cleavage in conventional assays. (A) EMSA using as a probe 5 nM 23-RSS doubly 5′ end labeled with FAM (circle) at its nonamer end and with ³²P (star) at its heptamer end. Partner RSSs were omitted (lane 2) or added at 1× or 5× stoichiometry as indicated above the lanes (TAMRA is indicated as a black oval). Lane 1, no protein; lanes 2 through 9, 65 nM RAG1, 125 nM RAG2, and 500 nM HMGB2. Only the portion of the gel containing the shifted complexes is shown. Two exposures are provided to allow clear visualization of both strong and weak bands. 12- and 23-RSSs are indicated by white and gray triangles, respectively. The SC and PC are indicated by arrows. + and −, presence or absence, respectively, of the indicated component. (B) Kinetics of RAG-mediated DNA cleavage using the same doubly labeled 23-RSS (5 nM) as that described in the legend to panel A. The RAG and HMGB (HMG) proteins and 12-RSS partner were added as indicated above the lanes, and cleavage was allowed to proceed for the times (min) indicated. The structure and stoichiometry of the three partner RSSs are indicated at the top, using symbols as described in the legend to panel A. Lanes 1, 6, and 10, no protein; all other lanes, 65 nM RAG1, 125 nM RAG2, and 500 nM HMGB2. Lane 2, no partner RSS. The input substrate and nicked and hairpin products can be visualized, as depicted schematically at the right.