Genomes and Genes
IMMUNOGLOBULIN AND T CELL RECEPTOR GENE ASSEMBLY
Principal Investigator: David Schatz
Abstract: V(D)J recombination is the site-specific recombination reaction that assembles the variable portions of immunoglobulin and T cell receptor genes. The reaction is essential for lymphocyte development and in many species is responsible for generating the diverse array of antigen receptor genes necessary for an effective immune response. Errors in the recombination process can lead to chromosomal translocations and hence to the development of human malignancies, particularly childhood leukemias. The causes of such translocations are thought to be improper targeting of the recombination machinery (DNA recognition errors), and the premature release of broken chromosomal ends from the recombination machinery before they have been properly joined (end release errors). Very little is known, however, about how such errors occur or what cellular processes act to prevent them. The proteins encoded by the recombination activating genes, RAG1 and RAG2, play central roles in the targeting of V(D)J recombination and in the efficient rejoining of the broken DNA ends. The RAG proteins are therefore likely to be crucial determinants of genomic stability in developing lymphocytes. The hypothesis underlying this application is that V(D)J recombination occurs within highly organized nucleoprotein structures whose integrity and specificity are in large part determined by RAG1 and RAG2. We propose to investigate the critical protein-protein and protein-DNA interactions involving RAG1 and RAG2 using conventional and recently developed, spectroscopic methods. A particular emphasis will be on understanding interaction surfaces and dynamic aspects of the reaction, especially conformational changes that occur when RAG1 interacts with RAG2 or with DNA. In addition, we will use biochemical and fluorescence methods to investigate the formation and structure of essential higher order "synaptic" complexes formed during V(D)J recombination. Finally, we have identified RAG1 mutants prone to end release errors in vitro, and will target these mutations to the endogenous murine RAG1 locus by homologous recombination. These mice will allow us to test the hypothesis that RAG-mediated stabilization of post-cleavage complexes is important for genome integrity and the prevention of lymphoid tumors. We will test the contribution of "gatekeeper" and "caretaker" genes to the suppression of such tumors and attempt to establish an animal model of lymphomagenesis. Overall, our experiments will address both the targeting and rejoining functions of the RAG proteins, with the long term goal of linking these fundamental activities to chromosomal translocations found in human malignancies.
Funding Period: 1992-04-01 - 2007-05-31
more information: NIH RePORT
- Cooperative recruitment of HMGB1 during V(D)J recombination through interactions with RAG1 and DNAAlicia J Little
Department of Immunobiology, Yale University School of Medicine, 300 Cedar Street, New Haven, CT 06511, USA
Nucleic Acids Res 41:3289-301. 2013..These findings also have implications for the order of events during RAG-DNA complex assembly and for the stabilization of sequence-specific and non-specific RAG1-DNA interactions...
- RAG and HMGB1 create a large bend in the 23RSS in the V(D)J recombination synaptic complexesMihai Ciubotaru
Department of Immunobiology, Yale University School of Medicine, 300 Cedar St, New Haven, CT 06511, USA
Nucleic Acids Res 41:2437-54. 2013..We propose that this large bend facilitates simultaneous recognition of the heptamer and nonamer, is critical for proper positioning of the active site and contributes to the 12/23 rule...
- Localized epigenetic changes induced by DH recombination restricts recombinase to DJH junctionsRamesh Subrahmanyam
Laboratory of Molecular Biology and Immunology, National Institute on Aging, US National Institutes of Health, Baltimore, Maryland, USA
Nat Immunol 13:1205-12. 2012..We propose that V(H) recombination is precise, because these changes did not extend to germline D(H) segments located 5' of the DJ(H) junction...
- The in vivo pattern of binding of RAG1 and RAG2 to antigen receptor lociYanhong Ji
Department of Immunobiology, Yale University School of Medicine, 300 Cedar Street, Box 208011, New Haven, CT 06520 8011, USA
Cell 141:419-31. 2010..We propose that recombination centers coordinate V(D)J recombination by providing discrete sites within which gene segments are captured for recombination...
- Structure of the RAG1 nonamer binding domain with DNA reveals a dimer that mediates DNA synapsisFang Fang Yin
Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, Connecticut, USA
Nat Struct Mol Biol 16:499-508. 2009..These findings reveal a previously unsuspected function for the NBD in DNA synapsis and have implications for the regulation of DNA binding and cleavage by RAG1 and RAG2...
- Ebf1-dependent control of the osteoblast and adipocyte lineagesDavid G T Hesslein
Department of Cell Biology, Yale University School of Medicine, New Haven, CT 06510, USA
Bone 44:537-46. 2009..Thus Ebf1-deficient mice appear to be a new model of lipodystrophy. Ebf1 is a rare example of a transcription factor that regulates both the osteoblast and adipocyte lineages similarly...
- Leaky severe combined immunodeficiency and aberrant DNA rearrangements due to a hypomorphic RAG1 mutationWilliam Giblin
Department of Internal Medicine, University of Michigan, Ann Arbor, MI 48109, USA
Blood 113:2965-75. 2009..Thus, our study provides in vivo evidence that implicates aberrant RAG1/2 activity in lymphoid tumor development and premature immunosenescence...
- The beyond 12/23 restriction is imposed at the nicking and pairing steps of DNA cleavage during V(D)J recombinationAnna H Drejer-Teel
Department of Genetics, Yale University School of Medicine, 330 Cedar St, New Haven, Connecticut 06510, USA
Mol Cell Biol 27:6288-99. 2007....
- Fluorescence resonance energy transfer analysis of recombination signal sequence configuration in the RAG1/2 synaptic complexMihai Ciubotaru
Howard Hughes Medical Institute and Department of Immunibiology, Yale University School of Medicine, New Haven, CT 06520 8011, USA
Mol Cell Biol 27:4745-58. 2007....
- Biochemistry of V(D)J recombinationD G Schatz
Section of Immunobiology, Howard Hughes Medical Institute, Yale University School of Medicine, New Haven, CT 06520 8011, USA
Curr Top Microbiol Immunol 290:49-85. 2005..We discuss how the RAG proteins interact with DNA and how coordinate cleavage of the DNA at two sites might be achieved. Finally, we consider the RAG proteins and V(D)J recombination from an evolutionary point of view...
- Mobilization of RAG-generated signal ends by transposition and insertion in vivoMonalisa Chatterji
Howard Hughes Medical Institute, Section of Immunobiology, Yale University School of Medicine, 300 Cedar Street, Box 208011, New Haven, CT 06520 8011, USA
Mol Cell Biol 26:1558-68. 2006..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...
- Origins of peripheral B cells in IL-7 receptor-deficient miceDavid G T Hesslein
Department of Cell Biology, Yale University School of Medicine, New Haven, CT 06519, USA
Mol Immunol 43:326-34. 2006..We conclude that the majority of splenic B cells in IL-7Ralpha-deficient mice originate from the bone marrow and not the fetal liver...