FIG. 4.

Detection of the PC by FRET. Donor and acceptor RSS substrates were incubated with RAG1 and HMGB2 but no RAG2 or with RAG1, RAG2, and HMGB2 (total RAG mix) for 10 or 20 min in an Mg²⁺ buffer as described in Materials and Methods. Steady-state emission spectra were recorded using an excitation wavelength of 492 nm, and the spectra determined by subtraction (to correct for background TAMRA emission) are shown (see Materials and Methods). (A) Steady-state emission spectra obtained using a 23-RSS labeled at its heptamer end with FAM and a 12-RSS substrate (fivefold molar excess) labeled at its nonamer end with TAMRA. Fluorescence I, fluorescence intensity expressed in arbitrary units. (B) Steady-state emission spectra obtained using a 23-RSS labeled at its heptamer end with FAM and a 12-RSS (fivefold molar excess) labeled at its heptamer end with TAMRA. Results for the no-RAG2 control and the total RAG mix (10-min time point) are shown, as is the spectrum obtained for the total RAG mix after the addition of EDTA to a final concentration of 17 mM (20-min time point). +, with; −, without. (C) FRET requires HMGB2. Using the same configuration of fluorophores described in the legend to panel B, spectra were recorded (in the absence of HMGB2) with RAG1 alone and with RAG1 and RAG2 at 10 and 20 min.