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Mol. Cell. Biol. doi:10.1128/MCB.00177-07
Copyright (c) 2007, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.

Fluorescence resonance energy transfer analysis of RSS configuration in the RAG1/2 synaptic complex

Howard Hughes Medical Institute, Yale University School of Medicine, Department of Immunobiology, 300 Cedar St., New Haven, CT 06510; Department of Medical Microbiology and Immunology, Creighton University Medical Center, Omaha, Nebraska 68178; University at Buffalo, The Department of Chemistry, Buffalo, NY 12640

^* To whom correspondence should be addressed. Email: david.schatz{at}yale.edu.

	Abstract

A critical step in V(D)J recombination is synapsis of complementary (12/23) recombination signal sequences (RSSs) by the RAG1/2 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 geometry of the RSSs is favored in the PC. To investigate this configuration further, we used fluorescence resonance energy transfer (FRET) to detect synapsis of fluorescently-labeled RSS oligonucleotides. FRET requires an appropriate 12/23 RSS pair, divalent metal ion, and HMGB1/2. Energy transfer between the RSSs was detected with all 12/23 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 using an in gel 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 23 RSSs are bent and cross one another, with implications for the organization of the RAG proteins and the DNA substrates at the time of cleavage.