Antigen receptor loci are composed of numerous variable (V), diversity (D), and joining (J) gene segments, each flanked by recombination signal sequences (RSSs). The V(D)J recombination reaction proceeds through RSS recognition and DNA cleavage steps making it possible for multiple DNA double strand breaks (DSBs) to be introduced at a single locus. Here we use ligation-mediated PCR to analyze DNA cleavage intermediates in thymocytes from mice with targeted RSS mutations at the endogenous TCRβ locus. We show that DNA cleavage does not occur at individual RSSs but rather must be coordinated between RSS pairs flanking gene segments that ultimately form coding joins. Coordination of the DNA cleavage step occurs over great distances in the chromosome and favors intra- over interchromosomal recombination. Furthermore, through several restrictions imposed on the generation of both nonpaired and paired DNA DSBs, this requirement promotes antigen receptor gene integrity and genomic stability in developing lymphocytes undergoing V(D)J recombination.
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4 February 2002
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January 28 2002
Restrictions Limiting the Generation of DNA Double Strand Breaks during Chromosomal V(D)J Recombination
Robert E. Tillman,
Robert E. Tillman
Washington University School of Medicine, Department of Pathology and Immunology, St. Louis, MO 63110
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Andrea L. Wooley,
Andrea L. Wooley
Washington University School of Medicine, Department of Pathology and Immunology, St. Louis, MO 63110
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Maureen M. Hughes,
Maureen M. Hughes
Washington University School of Medicine, Department of Pathology and Immunology, St. Louis, MO 63110
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Tara D. Wehrly,
Tara D. Wehrly
Washington University School of Medicine, Department of Pathology and Immunology, St. Louis, MO 63110
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Wojciech Swat,
Wojciech Swat
Washington University School of Medicine, Department of Pathology and Immunology, St. Louis, MO 63110
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Barry P. Sleckman
Barry P. Sleckman
Washington University School of Medicine, Department of Pathology and Immunology, St. Louis, MO 63110
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Robert E. Tillman
Washington University School of Medicine, Department of Pathology and Immunology, St. Louis, MO 63110
Andrea L. Wooley
Washington University School of Medicine, Department of Pathology and Immunology, St. Louis, MO 63110
Maureen M. Hughes
Washington University School of Medicine, Department of Pathology and Immunology, St. Louis, MO 63110
Tara D. Wehrly
Washington University School of Medicine, Department of Pathology and Immunology, St. Louis, MO 63110
Wojciech Swat
Washington University School of Medicine, Department of Pathology and Immunology, St. Louis, MO 63110
Barry P. Sleckman
Washington University School of Medicine, Department of Pathology and Immunology, St. Louis, MO 63110
Address correspondence to Barry P. Sleckman, Washington University School of Medicine, Department of Pathology and Immunology, 660 S. Euclid Ave., Campus Box 8118, St. Louis, MO 63110-1093. Phone: 314-747-8235; Fax: 314-362-4096; E-mail: [email protected]
*
Abbreviations used in this paper: DSB, double strand break; LMPCR, ligation-mediated PCR; RAG, recombinase activating gene; RSS, recombination signal sequence.
Received:
October 26 2001
Revision Received:
December 04 2001
Accepted:
December 12 2001
Online ISSN: 1540-9538
Print ISSN: 0022-1007
The Rockefeller University Press
2002
J Exp Med (2002) 195 (3): 309–316.
Article history
Received:
October 26 2001
Revision Received:
December 04 2001
Accepted:
December 12 2001
Citation
Robert E. Tillman, Andrea L. Wooley, Maureen M. Hughes, Tara D. Wehrly, Wojciech Swat, Barry P. Sleckman; Restrictions Limiting the Generation of DNA Double Strand Breaks during Chromosomal V(D)J Recombination . J Exp Med 4 February 2002; 195 (3): 309–316. doi: https://doi.org/10.1084/jem.20011803
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