fluorescence resonance energy transfer analysis of recombination signal sequence configuration in the rag12 synaptic complex a critical step in vdj recombination is the synapsis of complementary 1223 recombination signal sequences rsss by the rag1 and rag2 proteins to generate the paired complex pc using a facilitated ligation assay and substrates that vary the helical phasing of the rsss we provide evidence that one particular geometric configuration of the rsss is favored in the pc to investigate this configuration further we used fluorescent resonance energy transfer fret to detect the synapsis of fluorescently labeled rss oligonucleotides fret requires an appropriate 1223 rss pair a divalent metal ion and highmobilitygroup protein hmgb1 or hmgb2 energy transfer between the rsss was detected with all 1223 rss end positions of the fluorescent probes but was not detected when probes were placed on the two ends of the same rss energy transfer was confirmed to originate from the pc by using an ingel fret assay the results argue against a unique planar configuration of the rsss in the pc and are most easily accommodated by models in which synapsed 12 and 23rsss are bent and cross one another with implications for the organization of the rag proteins and the dna substrates at the time of cleavage