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 Kim, D. R.
Right arrow Articles by Oettinger, M. A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Kim, D. R.
Right arrow Articles by Oettinger, M. A.

 Previous Article  |  Next Article 

Mol Cell Biol, August 1998, p. 4679-4688, Vol. 18, No. 8
0270-7306/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.

Functional Analysis of Coordinated Cleavage in V(D)J Recombination

Deok Ryong Kim and Marjorie A. Oettinger*

Department of Molecular Biology, Massachusetts General Hospital, Boston, Massachusetts 02114

Received 4 March 1998/Returned for modification 13 April 1998/Accepted 8 May 1998

V(D)J recombination in vivo requires a pair of signals with distinct spacer elements of 12 and 23 bp that separate conserved heptamer and nonamer motifs. Cleavage in vitro by the RAG1 and RAG2 proteins can occur at individual signals when the reaction buffer contains Mn2+, but cleavage is restricted to substrates containing two signals when Mg2+ is the divalent cation. By using a novel V(D)J cleavage substrate, we show that while the RAG proteins alone establish a moderate preference for a 12/23 pair versus a 12/12 pair, a much stricter dependence of cleavage on the 12/23 signal pair is produced by the inclusion of HMG1 and competitor double-stranded DNA. The competitor DNA serves to inhibit the cleavage of substrates carrying a 12/12 or 23/23 pair, as well as the cutting at individual signals in 12/23 substrates. We show that a 23/33 pair is more efficiently recombined than a 12/33 pair, suggesting that the 12/23 rule can be generalized to a requirement for spacers that differ from each other by a single helical turn. Furthermore, we suggest that a fixed spatial orientation of signals is required for cleavage. In general, the same signal variants that can be cleaved singly can function under conditions in which a signal pair is required. However, a chemically modified substrate with one noncleavable signal enables us to show that formation of a functional cleavage complex is mechanistically separable from the cleavage reaction itself and that although cleavage requires a pair of signals, cutting does not have to occur simultaneously at both. The implications of these results are discussed with respect to the mechanism of V(D)J recombination and the generation of chromosomal translocations.

* Corresponding author. Mailing address: Department of Molecular Biology, Massachusetts General Hospital, Boston, MA 02114. Phone: (617) 726-5967. Fax: (617) 726-5949. E-mail: Oettinger{at}frodo.mgh.harvard.edu.

Mol Cell Biol, August 1998, p. 4679-4688, Vol. 18, No. 8
0270-7306/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.


This article has been cited by other articles:

  • Bischerour, J., Lu, C., Roth, D. B., Chalmers, R. (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: 5889-5899 [Abstract] [Full Text]  
  • Wright, S. M., Woo, Y. H., Alley, T. L., Shirley, B.-J., Akeson, E. C., Snow, K. J., Maas, S. A., Elwell, R. L., Foreman, O., Mills, K. D. (2009). Complex Oncogenic Translocations with Gene Amplification Are Initiated by Specific DNA Breaks in Lymphocytes. Cancer Res. 69: 4454-4460 [Abstract] [Full Text]  
  • Nishihara, T., Nagawa, F., Imai, T., Sakano, H. (2008). RAG-Heptamer Interaction in the Synaptic Complex Is a Crucial Biochemical Checkpoint for the 12/23 Recombination Rule. J. Biol. Chem. 283: 4877-4885 [Abstract] [Full Text]  
  • Grundy, G. J., Hesse, J. E., Gellert, M. (2007). Requirements for DNA hairpin formation by RAG1/2. Proc. Natl. Acad. Sci. USA 104: 3078-3083 [Abstract] [Full Text]  
  • Nagawa, F., Hirose, S., Nishizumi, H., Nishihara, T., Sakano, H. (2004). Joining Mutants of RAG1 and RAG2 that Demonstrate Impaired Interactions with the Coding-end DNA. J. Biol. Chem. 279: 38360-38368 [Abstract] [Full Text]  
  • Sawchuk, D. J., Mansilla-Soto, J., Alarcon, C., Singha, N. C., Langen, H., Bianchi, M. E., Lees-Miller, S. P., Nussenzweig, M. C., Cortes, P. (2004). Ku70/Ku80 and DNA-dependent Protein Kinase Catalytic Subunit Modulate RAG-mediated Cleavage: IMPLICATIONS FOR THE ENFORCEMENT OF THE 12/23 RULE. J. Biol. Chem. 279: 29821-29831 [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]  
  • Montalbano, A., Ogwaro, K. M., Tang, A., Matthews, A. G. W., Larijani, M., Oettinger, M. A., Feeney, A. J. (2003). V(D)J Recombination Frequencies Can Be Profoundly Affected by Changes in the Spacer Sequence. J. Immunol. 171: 5296-5304 [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]  
  • Yu, K., Taghva, A., Lieber, M. R. (2002). The Cleavage Efficiency of the Human Immunoglobulin Heavy Chain VH Elements by the RAG Complex. IMPLICATIONS FOR THE IMMUNE REPERTOIRE. J. Biol. Chem. 277: 5040-5046 [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]  
  • Neiditch, M. B., Lee, G. S., Landree, M. A., Roth, D. B. (2001). RAG Transposase Can Capture and Commit to Target DNA before or after Donor Cleavage. Mol. Cell. Biol. 21: 4302-4310 [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]  
  • Williams, C. J., Grandal, I., Vesprini, D. J., Wojtyra, U., Danska, J. S., Guidos, C. J. (2001). Irradiation Promotes V(D)J Joining and RAG-Dependent Neoplastic Transformation in SCID T-Cell Precursors. Mol. Cell. Biol. 21: 400-413 [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]  
  • Huye, L. E., Roth, D. B. (2000). Differential requirements for cis and trans V(D)J cleavage: effects of substrate length. Nucleic Acids Res 28: 4903-4911 [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]  
  • 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]  
  • Yoshida, T., Tsuboi, A., Ishiguro, K.-i., Nagawa, F., Sakano, H. (2000). The DNA-bending protein, HMG1, is required for correct cleavage of 23 bp recombination signal sequences by recombination activating gene proteins in vitro. Int Immunol 12: 721-729 [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]  
  • Kim, D. R., Dai, Y., Mundy, C. L., Yang, W., Oettinger, M. A. (1999). Mutations of acidic residues in RAG1 define the active site of the V(D)J recombinase. Genes Dev. 13: 3070-3080 [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]  
  • Bailin, T., Mo, X., Sadofsky, M. J. (1999). A RAG1 and RAG2 Tetramer Complex Is Active in Cleavage in V(D)J Recombination. Mol. Cell. Biol. 19: 4664-4671 [Abstract] [Full Text]  
  • Akamatsu, Y., Oettinger, M. A. (1998). Distinct Roles of RAG1 and RAG2 in Binding the V(D)J Recombination Signal Sequences. Mol. Cell. Biol. 18: 4670-4678 [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»