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Molecular and Cellular Biology, February 2006, p. 1558-1568, Vol. 26, No. 4
0270-7306/06/$08.00+0     doi:10.1128/MCB.26.4.1558-1568.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Mobilization of RAG-Generated Signal Ends by Transposition and Insertion In Vivo

Monalisa Chatterji, Chia-Lun Tsai, and David G. Schatz^*

Howard Hughes Medical Institute, Section of Immunobiology, Yale University School of Medicine, 300 Cedar Street, Box 208011, New Haven, Connecticut 06520-8011

Received 20 August 2005/ Returned for modification 21 September 2005/ Accepted 23 November 2005

In addition to their essential roles in V(D)J recombination, the RAG proteins have been found to catalyze transposition in vitro, but it has been difficult to demonstrate transposition by the RAG proteins in vivo in vertebrate cells. As genomic instability and chromosomal translocations are common outcomes of transposition in other species, it is critical to understand if the RAG proteins behave as a transposase in vertebrate cells. To facilitate this, we have developed an episome-based assay to detect products of RAG-mediated transposition in the human embryonic kidney cell line 293T. Transposition events into the target episome, accompanied by characteristic target site duplications, were detected at a low frequency using RAG1 and either truncated "core" RAG2 or full-length RAG2. More frequently, insertion of the RAG-generated signal end fragment into the target was accompanied by deletions or more complex rearrangements, and our data indicate that these events occur by a mechanism that is distinct from transposition. An assay to detect transposition from an episome into the human genome failed to detect bona fide transposition events but instead yielded chromosome deletion and translocation events involving the signal end fragment mobilized by the RAG proteins. These assays provide a means of assessing RAG-mediated transposition in vivo, and our findings provide insight into the potential for the products of RAG-mediated DNA cleavage to cause genome instability.

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* Corresponding author. Mailing address: Howard Hughes Medical Institute, Section of Immunobiology, Yale University School of Medicine, 300 Cedar Street, Box 208011, New Haven, CT 06520-8011. Phone: (203) 737-2255. Fax: (203) 785-3855. E-mail: david.schatz{at}yale.edu.

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

Present address: Howard Hughes Medical Institute, Department of Molecular Biology, Wellman 9, Massachusetts General Hospital, Boston, MA 02114.

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Molecular and Cellular Biology, February 2006, p. 1558-1568, Vol. 26, No. 4
0022-538X/06/$08.00+0     doi:10.1128/MCB.26.4.1558-1568.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

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