This Article
Right arrow Full Text
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Swanson, P. C.
Right arrow Articles by Desiderio, S.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Swanson, P. C.
Right arrow Articles by Desiderio, S.

 Previous Article  |  Next Article 

Molecular and Cellular Biology, May 1999, p. 3674-3683, Vol. 19, No. 5
0270-7306/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.

RAG-2 Promotes Heptamer Occupancy by RAG-1 in the Assembly of a V(D)J Initiation Complexdagger

Patrick C. Swanson and Stephen Desiderio*

Department of Molecular Biology and Genetics and Howard Hughes Medical Institute, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205

Received 28 December 1998/Returned for modification 3 February 1999/Accepted 12 February 1999

V(D)J recombination occurs at recombination signal sequences (RSSs) containing conserved heptamer and nonamer elements. RAG-1 and RAG-2 initiate recombination by cleaving DNA between heptamers and antigen receptor coding segments. RAG-1 alone contacts the nonamer but interacts weakly, if at all, with the heptamer. RAG-2 by itself has no DNA-binding activity but promotes heptamer occupancy in the presence of RAG-1; how RAG-2 collaborates with RAG-1 has been poorly understood. Here we examine the composition of RAG-RSS complexes and the relative contributions of RAG-1 and RAG-2 to heptamer binding. RAG-1 exists as a dimer in complexes with an isolated RSS bearing a 12-bp spacer, regardless of whether RAG-2 is present; only a single subunit of RAG-1, however, participates in nonamer binding. In contrast, multimeric RAG-2 is not detectable by electrophoretic mobility shift assays in complexes containing both RAG proteins. DNA-protein photo-cross-linking demonstrates that heptamer contacts, while enhanced by RAG-2, are mediated primarily by RAG-1. RAG-2 cross-linking, while less efficient than that of RAG-1, is detectable near the heptamer-coding junction. These observations provide evidence that RAG-2 alters the conformation or orientation of RAG-1, thereby stabilizing interactions of RAG-1 with the heptamer, and suggest that both proteins interact with the RSS near the site of cleavage.

* Corresponding author. Mailing address: Department of Molecular Biology and Genetics and Howard Hughes Medical Institute, The Johns Hopkins University School of Medicine, Baltimore, MD 21205. Phone: (410) 955-4735. Fax: (410) 955-9124. E-mail: sdesider{at}jhmi.edu.

dagger We dedicate this paper to the memory of our friend and colleague Eugenia Spanopoulou.

Molecular and Cellular Biology, May 1999, p. 3674-3683, Vol. 19, No. 5
0270-7306/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.


This article has been cited by other articles:

  • Lu, C. P., Posey, J. E., Roth, D. B. (2008). Understanding how the V(D)J recombinase catalyzes transesterification: distinctions between DNA cleavage and transposition. Nucleic Acids Res 36: 2864-2873 [Abstract] [Full Text]  
  • Bergeron, S., Madathiparambil, T., Swanson, P. C. (2005). Both High Mobility Group (HMG)-boxes and the Acidic Tail of HMGB1 Regulate Recombination-activating Gene (RAG)-mediated Recombination Signal Synapsis and Cleavage in Vitro. J. Biol. Chem. 280: 31314-31324 [Abstract] [Full Text]  
  • Ciubotaru, M., Schatz, D. G. (2004). Synapsis of Recombination Signal Sequences Located in cis and DNA Underwinding in V(D)J Recombination. Mol. Cell. Biol. 24: 8727-8744 [Abstract] [Full Text]  
  • De, P., Peak, M. M., Rodgers, K. K. (2004). DNA Cleavage Activity of the V(D)J Recombination Protein RAG1 Is Autoregulated. Mol. Cell. Biol. 24: 6850-6860 [Abstract] [Full Text]  
  • Ko, J. E., Kim, C. W., Kim, D. R. (2004). Amino Acid Residues in RAG1 Responsible for the Interaction with RAG2 during the V(D)J Recombination Process. J. Biol. Chem. 279: 7715-7720 [Abstract] [Full Text]  
  • Swanson, P. C., Volkmer, D., Wang, L. (2004). Full-length RAG-2, and Not Full-length RAG-1, Specifically Suppresses RAG-mediated Transposition but Not Hybrid Joint Formation or Disintegration. J. Biol. Chem. 279: 4034-4044 [Abstract] [Full Text]  
  • Hughes, M. M., Tillman, R. E., Wehrly, T. D., White, J. M., Sleckman, B. P. (2003). The B12/23 Restriction Is Critically Dependent on Recombination Signal Nonamer and Spacer Sequences. J. Immunol. 171: 6604-6610 [Abstract] [Full Text]  
  • Ross, A. E., Vuica, M., Desiderio, S. (2003). Overlapping Signals for Protein Degradation and Nuclear Localization Define a Role for Intrinsic RAG-2 Nuclear Uptake in Dividing Cells. Mol. Cell. Biol. 23: 5308-5319 [Abstract] [Full Text]  
  • Peak, M. M., Arbuckle, J. L., Rodgers, K. K. (2003). The Central Domain of Core RAG1 Preferentially Recognizes Single-stranded Recombination Signal Sequence Heptamer. J. Biol. Chem. 278: 18235-18240 [Abstract] [Full Text]  
  • Godderz, L. J., Rahman, N. S., Risinger, G. M., Arbuckle, J. L., Rodgers, K. K. (2003). Self-association and conformational properties of RAG1: implications for formation of the V(D)J recombinase. Nucleic Acids Res 31: 2014-2023 [Abstract] [Full Text]  
  • Ciubotaru, M., Ptaszek, L. M., Baker, G. A., Baker, S. N., Bright, F. V., Schatz, D. G. (2003). RAG1-DNA Binding in V(D)J Recombination. SPECIFICITY AND DNA-INDUCED CONFORMATIONAL CHANGES REVEALED BY FLUORESCENCE AND CD SPECTROSCOPY. J. Biol. Chem. 278: 5584-5596 [Abstract] [Full Text]  
  • Swanson, P. C. (2002). A RAG-1/RAG-2 Tetramer Supports 12/23-Regulated Synapsis, Cleavage, and Transposition of V(D)J Recombination Signals. Mol. Cell. Biol. 22: 7790-7801 [Abstract] [Full Text]  
  • Nagawa, F., Kodama, M., Nishihara, T., Ishiguro, K.-i., Sakano, H. (2002). Footprint Analysis of Recombination Signal Sequences in the 12/23 Synaptic Complex of V(D)J Recombination. Mol. Cell. Biol. 22: 7217-7225 [Abstract] [Full Text]  
  • Huye, L. E., Purugganan, M. M., Jiang, M.-M., Roth, D. B. (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: 3460-3473 [Abstract] [Full Text]  
  • Swanson, P. C. (2002). Fine Structure and Activity of Discrete RAG-HMG Complexes on V(D)J Recombination Signals. Mol. Cell. Biol. 22: 1340-1351 [Abstract] [Full Text]  
  • Mundy, C. L., Patenge, N., Matthews, A. G. W., Oettinger, M. A. (2002). Assembly of the RAG1/RAG2 Synaptic Complex. Mol. Cell. Biol. 22: 69-77 [Abstract] [Full Text]  
  • Landree, M. A., Kale, S. B., Roth, D. B. (2001). Functional Organization of Single and Paired V(D)J Cleavage Complexes. Mol. Cell. Biol. 21: 4256-4264 [Abstract] [Full Text]  
  • Li, W., Chang, F.-C., Desiderio, S. (2001). RAG-1 Mutations Associated with B-Cell-Negative SCID Dissociate the Nicking and Transesterification Steps of V(D)J Recombination. Mol. Cell. Biol. 21: 3935-3946 [Abstract] [Full Text]  
  • Sadofsky, M. J. (2001). The RAG proteins in V(D)J recombination: more than just a nuclease. Nucleic Acids Res 29: 1399-1409 [Abstract] [Full Text]  
  • Mo, X., Bailin, T., Sadofsky, M. J. (2001). A C-Terminal Region of RAG1 Contacts the Coding DNA during V(D)J Recombination. Mol. Cell. Biol. 21: 2038-2047 [Abstract] [Full Text]  
  • Swanson, P. C. (2001). The DDE Motif in RAG-1 Is Contributed in trans to a Single Active Site That Catalyzes the Nicking and Transesterification Steps of V(D)J Recombination. Mol. Cell. Biol. 21: 449-458 [Abstract] [Full Text]  
  • Kale, S. B., Landree, M. A., Roth, D. B. (2001). Conditional RAG-1 Mutants Block the Hairpin Formation Step of V(D)J Recombination. Mol. Cell. Biol. 21: 459-466 [Abstract] [Full Text]  
  • Yu, K., Lieber, M. R. (2000). The Nicking Step in V(D)J Recombination Is Independent of Synapsis: Implications for the Immune Repertoire. Mol. Cell. Biol. 20: 7914-7921 [Abstract] [Full Text]  
  • Gomez, C. A., Ptaszek, L. M., Villa, A., Bozzi, F., Sobacchi, C., Brooks, E. G., Notarangelo, L. D., Spanopoulou, E., Pan, Z. Q., Vezzoni, P., Cortes, P., Santagata, S. (2000). Mutations in Conserved Regions of the Predicted RAG2 Kelch Repeats Block Initiation of V(D)J Recombination and Result in Primary Immunodeficiencies. Mol. Cell. Biol. 20: 5653-5664 [Abstract] [Full Text]  
  • Aidinis, V., Dias, D. C., Gomez, C. A., Bhattacharyya, D., Spanopoulou, E., Santagata, S. (2000). Definition of Minimal Domains of Interaction Within the Recombination-Activating Genes 1 and 2 Recombinase Complex. J. Immunol. 164: 5826-5832 [Abstract] [Full Text]  
  • Corneo, B., Moshous, D., Callebaut, I., de Chasseval, R., Fischer, A., de Villartay, J.-P. (2000). Three-dimensional Clustering of Human RAG2 Gene Mutations in Severe Combined Immune Deficiency. J. Biol. Chem. 275: 12672-12675 [Abstract] [Full Text]  
  • Mo, X., Bailin, T., Noggle, S., Sadofsky, M. J. (2000). A highly ordered structure in V(D)J recombination cleavage complexes is facilitated by HMG1. Nucleic Acids Res 28: 1228-1236 [Abstract] [Full Text]  
  • Yu, K., Lieber, M. R. (1999). Mechanistic Basis for Coding End Sequence Effects in the Initiation of V(D)J Recombination. Mol. Cell. Biol. 19: 8094-8102 [Abstract] [Full Text]  
  • Landree, M. A., Wibbenmeyer, J. A., Roth, D. B. (1999). Mutational analysis of RAG1 and RAG2 identifies three catalytic amino acids in RAG1 critical for both cleavage steps of V(D)J recombination. Genes Dev. 13: 3059-3069 [Abstract] [Full Text]  
  • Aidinis, V., Bonaldi, T., Beltrame, M., Santagata, S., Bianchi, M. E., Spanopoulou, E. (1999). The RAG1 Homeodomain Recruits HMG1 and HMG2 To Facilitate Recombination Signal Sequence Binding and To Enhance the Intrinsic DNA-Bending Activity of RAG1-RAG2. Mol. Cell. Biol. 19: 6532-6542 [Abstract] [Full Text]  
  • Arbuckle, J. L., Fauss, L. J., Simpson, R., Ptaszek, L. M., Rodgers, K. K. (2001). Identification of Two Topologically Independent Domains in RAG1 and Their Role in Macromolecular Interactions Relevant to V(D)J Recombination. J. Biol. Chem. 276: 37093-37101 [Abstract] [Full Text]  


Copyright © 2009 by the American Society for Microbiology.   For an alternate route to Journals.ASM.org, visit: http://intl-journals.asm.org | More Info»