Figure 6.
Detection of nonamer synapsis using FRET. (a) Diagram showing the positions of fluorophore labeling in the A, B, and C pairs of 12/23 RSS FRET substrates. TAMRA (pink circle); FAM (blue circle); CF, coding flank. (b) Representative corrected emission spectra obtained with the A substrate pair (left panel) or nonspecific (nsp) substrates, which are identical to the A substrates except that the nonamer and heptamer elements have been mutated in both the 12 and 23 RSSs (right panel). In each case, the FAM-labeled substrate was incubated with a 4-fold molar excess of either a TAMRA-labeled (pink spectrum) or unlabeled (blue, control spectrum) partner substrate together with RAG1/RAG2/HMGB1 in a buffer containing 5 mM MgCl2. Background intrinsic TAMRA fluorescence has been subtracted from both pink spectra. Donor quenching and TAMRA sensitization are observed only with the consensus A substrates.