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Molecular and Cellular Biology, July 2007, p. 4745-4758, Vol. 27, No. 13
0270-7306/07/$08.00+0     doi:10.1128/MCB.00177-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Fluorescence Resonance Energy Transfer Analysis of Recombination Signal Sequence Configuration in the RAG1/2 Synaptic Complex ^,

Mihai Ciubotaru,¹ Aleksei N. Kriatchko,² Patrick C. Swanson,² Frank V. Bright,³ and David G. Schatz^1*

Howard Hughes Medical Institute and Department of Immunobiology, Yale University School of Medicine, 300 Cedar St., New Haven, Connecticut 06510,¹ Department of Medical Microbiology and Immunology, Creighton University Medical Center, Omaha, Nebraska 68178,² Department of Chemistry, University at Buffalo, Buffalo, New York 12640³

Received 30 January 2007/ Returned for modification 5 April 2007/ Accepted 19 April 2007

A critical step in V(D)J recombination is the synapsis of complementary (12/23) 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 12/23 RSS pair, a divalent metal ion, and high-mobility-group protein HMGB1 or HMGB2. 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 by 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.

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* Corresponding author. Mailing address: Howard Hughes Medical Institute, Yale University School of Medicine, Department of Immunobiology, Box 208011, New Haven, CT, 06520-8011. Phone: (203) 737-2255. Fax: (203) 785-3855. E-mail: david.schatz{at}yale.edu

Published ahead of print on 30 April 2007.

Supplemental material for this article may be found at http://mcb.asm.org/.

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Molecular and Cellular Biology, July 2007, p. 4745-4758, Vol. 27, No. 13
0270-7306/07/$08.00+0     doi:10.1128/MCB.00177-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

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