The specialized recombination system responsible for assembling the variable regions of immunoglobulin and T cell receptor genes from germline gene segments is the principal mechanism for generating a diverse set of antigen receptors. This V(D)J recombination reaction is essential for the proper development of the immune system. Impairment of recombination, as in mutant mice with severe combined immunodeficiency (scid) results in a virtual absence of mature B and T lymphocytes. In addition to its crucial role in lymphocyte development, V(D)J recombination is responsible for aberrant rearrangements that result in lymphoid neoplasms. Therefore, understanding the mechanism of V(D)J recombination is important for understanding both normal development of the immune system and for determining the molecular basis of oncogenic recombination events. Recent work has demonstrated that recombination involves double-strand DNA breaks at recombination signal sequences, resulting in flush breaks at signal ends and covalently sealed (hairpin) coding ends. These broken molecules are likely to be intermediates in recombination, and their identification supports a hairpin model for V(D)J recombination. This project will address several critical questions about the recombination mechanism using sensitive tools to characterize the structure of hairpin termini and products of hairpin opening generated in vivo in mouse thymocytes. Experiments will also test whether the hairpin opening reaction is qualitatively or quantitatively affected by the scid mutation, as previously proposed. Additional studies will probe early steps in the recombination mechanism, including the potential role of complex formation involving pairs of recombination signals, and will address the molecular basis of the requirement for a pair of signals of different spacer lengths (the """"""""12-23"""""""" rule). The functional organization of the recombination signals will also be examined. Proposed studies will illuminate basic features of the V(D)J recombination mechanism and its regulation, and will further define the molecular basis of the scid recombination defect. This knowledge will increase our understanding of how certain human DNA repair disorders deregulate V(D)J recombination, resulting in an increased frequency of oncogenic chromosome translocations, and will provide insight into the molecular basis of human immunodeficiencies.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI036420-04
Application #
2413688
Study Section
Immunobiology Study Section (IMB)
Project Start
1994-08-01
Project End
1999-04-30
Budget Start
1997-05-01
Budget End
1998-04-30
Support Year
4
Fiscal Year
1997
Total Cost
Indirect Cost
Name
Baylor College of Medicine
Department
Microbiology/Immun/Virology
Type
Schools of Medicine
DUNS #
074615394
City
Houston
State
TX
Country
United States
Zip Code
77030
Bischerour, Julien; Lu, Catherine; Roth, David B et al. (2009) Base flipping in V(D)J recombination: insights into the mechanism of hairpin formation, the 12/23 rule, and the coordination of double-strand breaks. Mol Cell Biol 29:5889-99
Wong, Serre-Yu; Lu, Catherine P; Roth, David B (2008) A RAG1 mutation found in Omenn syndrome causes coding flank hypersensitivity: a novel mechanism for antigen receptor repertoire restriction. J Immunol 181:4124-30
Lu, Catherine P; Posey, Jennifer E; Roth, David B (2008) Understanding how the V(D)J recombinase catalyzes transesterification: distinctions between DNA cleavage and transposition. Nucleic Acids Res 36:2864-73
Lu, Catherine P; Sandoval, Hector; Brandt, Vicky L et al. (2006) Amino acid residues in Rag1 crucial for DNA hairpin formation. Nat Struct Mol Biol 13:1010-5
Posey, Jennifer E; Pytlos, Malgorzata J; Sinden, Richard R et al. (2006) Target DNA structure plays a critical role in RAG transposition. PLoS Biol 4:e350
Sandor, Zoltan; Calicchio, Monica L; Sargent, R Geoffrey et al. (2004) Distinct requirements for Ku in N nucleotide addition at V(D)J- and non-V(D)J-generated double-strand breaks. Nucleic Acids Res 32:1866-73
Lee, Gregory S; Neiditch, Matthew B; Salus, Sandra S et al. (2004) RAG proteins shepherd double-strand breaks to a specific pathway, suppressing error-prone repair, but RAG nicking initiates homologous recombination. Cell 117:171-84
Neiditch, Matthew B; Lee, Gregory S; Huye, Leslie E et al. (2002) The V(D)J recombinase efficiently cleaves and transposes signal joints. Mol Cell 9:871-8
Thai, To-Ha; Purugganan, Mary M; Roth, David B et al. (2002) Distinct and opposite diversifying activities of terminal transferase splice variants. Nat Immunol 3:457-62
Huye, Leslie E; Purugganan, Mary M; Jiang, Ming-Ming et al. (2002) Mutational analysis of all conserved basic amino acids in RAG-1 reveals catalytic, step arrest, and joining-deficient mutants in the V(D)J recombinase. Mol Cell Biol 22:3460-73

Showing the most recent 10 out of 35 publications