RAG1 and RAG2 Form a Stable Postcleavage Synaptic Complex with DNA Containing Signal Ends in V(D)J Recombination

During V(D)J recombination, RAG1 and RAG2 cleave DNA adjacent to highly conserved recombination signals, but nothing is known about the protein-DNA complexes that exist after cleavage. Using a properly regulated in vitro V(D)J cleavage system, together with nuclease sensitivity, mobility shift, and immunoprecipitation experiments, we provide evidence that a stable complex is formed postcleavage between synapsed recombination signals. This complex includes the proteins RAG1, RAG2, HMG-1 or the closely related HMG-2 protein, and the components of the DNA-dependent protein kinase. The existence of such a stable complex explains a number of in vivo observations and suggests that remodeling of postcleavage synaptic complexes is an important step in the resolution of signal ends in V(D)J recombination.

[1]  D. Schatz,et al.  T-cell receptor alpha locus V(D)J recombination by-products are abundant in thymocytes and mature T cells , 1996, Molecular and cellular biology.

[2]  D. Schatz,et al.  RAG-1 and RAG-2, adjacent genes that synergistically activate V(D)J recombination. , 1990, Science.

[3]  D. Baltimore,et al.  In vitro V(D)J recombination: signal joint formation. , 1996, Proceedings of the National Academy of Sciences of the United States of America.

[4]  D. V. van Gent,et al.  Similarities Between Initiation of V(D)J Recombination and Retroviral Integration , 1996, Science.

[5]  F. Alt,et al.  The XRCC4 gene encodes a novel protein involved in DNA double-strand break repair and V(D)J recombination , 1995, Cell.

[6]  I. Pastan,et al.  Pseudomonas exotoxin fusion proteins are potent immunogens for raising antibodies against P-glycoprotein. , 1991, BioTechniques.

[7]  Sandro Santagata,et al.  The Homeodomain Region of Rag-1 Reveals the Parallel Mechanisms of Bacterial and V(D)J Recombination , 1996, Cell.

[8]  G K Lewis,et al.  Isolation of monoclonal antibodies specific for human c-myc proto-oncogene product , 1985, Molecular and cellular biology.

[9]  I. Vancurova,et al.  A DNA-activated protein kinase from HeLa cell nuclei , 1990, Molecular and cellular biology.

[10]  T. Wirth,et al.  High mobility group protein 2 functionally interacts with the POU domains of octamer transcription factors. , 1995, The EMBO journal.

[11]  Molly A Bogue,et al.  Ku86-Deficient Mice Exhibit Severe Combined Immunodeficiency and Defective Processing of V(D)J Recombination Intermediates , 1996, Cell.

[12]  T. Baker,et al.  Disassembly of the Mu transposase tetramer by the ClpX chaperone. , 1995, Genes & development.

[13]  Naomi Rosenberg,et al.  The defect in murine severe combined immune deficiency: Joining of signal sequences but not coding segments in V(D)J recombination , 1988, Cell.

[14]  D. Weaver,et al.  What to do at an end: DNA double-strand-break repair. , 1995, Trends in genetics : TIG.

[15]  S. M. Lewis,et al.  Cutting and closing without recombination in V(D)J joining. , 1991, The EMBO journal.

[16]  David Baltimore,et al.  The V(D)J recombination activating gene, RAG-1 , 1989, Cell.

[17]  David G. Schatz,et al.  Initiation of V(D)J recombination in vitro obeying the 12/23 rule , 1996, Nature.

[18]  M. Simon,et al.  Host protein requirements for in vitro site-specific DNA inversion , 1986, Cell.

[19]  R. Reeves,et al.  High-mobility-group chromosomal proteins: architectural components that facilitate chromatin function. , 1996, Progress in nucleic acid research and molecular biology.

[20]  G. Chaconas,et al.  DNA transposition: jumping gene machine, some assembly required. , 1996 .

[21]  S. Lewis,et al.  The mechanism of V(D)J joining: lessons from molecular, immunological, and comparative analyses. , 1994, Advances in immunology.

[22]  D. Schatz,et al.  Identification of V(D)J recombination coding end intermediates in normal thymocytes. , 1997, Journal of molecular biology.

[23]  D. Roth,et al.  Characterization of broken DNA molecules associated with V(D)J recombination. , 1993, Proceedings of the National Academy of Sciences of the United States of America.

[24]  G. Wahl,et al.  Sensitivity and selectivity of the DNA damage sensor responsible for activating p53-dependent G1 arrest. , 1996, Proceedings of the National Academy of Sciences of the United States of America.

[25]  D. Ramsden,et al.  Formation and resolution of double-strand break intermediates in V(D)J rearrangement. , 1995, Genes & development.

[26]  K. Mizuuchi,et al.  Transpositional recombination: mechanistic insights from studies of mu and other elements. , 1992, Annual review of biochemistry.

[27]  R. C. Johnson,et al.  The nonspecific DNA-binding and -bending proteins HMG1 and HMG2 promote the assembly of complex nucleoprotein structures. , 1993, Genes & development.

[28]  D. Schatz.  V(D)J recombination moves in vitro. , 1997, Seminars in immunology.

[29]  M. Gellert,et al.  The mechanism of antigen receptor gene assembly , 1989, Cell.

[30]  M. Gellert,et al.  A Stable RAG1–RAG2–DNA Complex That Is Active in V(D)J Cleavage , 1997, Cell.

[31]  D. Ward,et al.  Mammalian ubiquitin-conjugating enzyme Ubc9 interacts with Rad51 recombination protein and localizes in synaptonemal complexes. , 1996, Proceedings of the National Academy of Sciences of the United States of America.

[32]  Christina A. Cuomo,et al.  Cleavage at a V(D)J recombination signal requires only RAG1 and RAG2 proteins and occurs in two steps , 1995, Cell.

[33]  P. Jeggo,et al.  DNA double-strand break repair and V(D)J recombination: involvement of DNA-PK. , 1995, Trends in biochemical sciences.

[34]  D. Schatz,et al.  rag-1 and rag-2 are components of a high-molecular-weight complex, and association of rag-2 with this complex is rag-1 dependent , 1995, Molecular and cellular biology.

[35]  D. Ramsden,et al.  Initiation of V(D)J recombination in a cell-free system , 1995, Cell.

[36]  J. Danska,et al.  V(D)J recombination activates a p53-dependent DNA damage checkpoint in scid lymphocyte precursors. , 1996, Genes & development.

[37]  M. Bustin.  Preparation and application of immunological probes for nucleosomes. , 1989, Methods in enzymology.

[38]  F. Alt,et al.  Impairment of V(D)J recombination in double-strand break repair mutants. , 1993, Science.

[39]  Eugenia Spanopoulou,et al.  RAG1 Mediates Signal Sequence Recognition and Recruitment of RAG2 in V(D)J Recombination , 1996, Cell.

[40]  H. Nakai,et al.  ClpX protein of Escherichia coli activates bacteriophage Mu transposase in the strand transfer complex for initiation of Mu DNA synthesis. , 1996, The EMBO journal.

[41]  D. Schatz.  V(D)J recombination moves in vitro. , 1997, Seminars in immunology.

[42]  M. Lieber,et al.  V(D)J recombination in mammalian cell mutants defective in DNA double-strand break repair , 1993, Molecular and cellular biology.

[43]  G. Chaconas,et al.  DNA transposition: Assembly of a jumping gene machine , 1996, Current Biology.

[44]  J. Menetski,et al.  V(D)J recombination in mouse thymocytes: Double-strand breaks near T cell receptor δ rearrangement signals , 1992, Cell.

[45]  Dale A Ramsden,et al.  The RAG1 and RAG2 Proteins Establish the 12/23 Rule in V(D)J Recombination , 1996, Cell.

[46]  D. Edwards,et al.  The DNA-bending protein HMG-1 enhances progesterone receptor binding to its target DNA sequences , 1994, Molecular and cellular biology.

[47]  J. Menetski,et al.  V(D)J recombination in mouse thymocytes: double-strand breaks near T cell receptor delta rearrangement signals. , 1992, Cell.

[48]  M. Nussenzweig,et al.  Requirement for Ku80 in growth and immunoglobulin V(D)J recombination , 1996, Nature.

[49]  H. Nash,et al.  Bending and supercoiling of DNA at the attachment site of bacteriophage lambda. , 1990, Trends in biochemical sciences.