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

 Previous Article  |  Next Article 

Molecular and Cellular Biology, June 2005, p. 4413-4425, Vol. 25, No. 11
0270-7306/05/$08.00+0     doi:10.1128/MCB.25.11.4413-4425.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Determinants of HMGB Proteins Required To Promote RAG1/2-Recombination Signal Sequence Complex Assembly and Catalysis during V(D)J Recombination

Yan Dai,^1, Ben Wong,^2, Yi-Meng Yen,² Marjorie A. Oettinger,^1* Jongbum Kwon,^1, and Reid C. Johnson^2*

Department of Molecular Biology, Massachusetts General Hospital, Boston, Massachusetts 02114,¹ Department of Biological Chemistry, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, California 90095-1737²

Received 16 January 2005/ Returned for modification 15 February 2005/ Accepted 1 March 2005

Efficient assembly of RAG1/2-recombination signal sequence (RSS) DNA complexes that are competent for V(D)J cleavage requires the presence of the nonspecific DNA binding and bending protein HMGB1 or HMGB2. We find that either of the two minimal DNA binding domains of HMGB1 is effective in assembling RAG1/2-RSS complexes on naked DNA and stimulating V(D)J cleavage but that both domains are required for efficient activity when the RSS is incorporated into a nucleosome. The single-domain HMGB protein from Saccharomyces cerevisiae, Nhp6A, efficiently assembles RAG1/2 complexes on naked DNA; however, these complexes are minimally competent for V(D)J cleavage. Nhp6A forms much more stable DNA complexes than HMGB1, and a variety of mutations that destabilize Nhp6A binding to bent microcircular DNA promote increased V(D)J cleavage. One of the two DNA bending wedges on Nhp6A and the analogous phenylalanine wedge at the DNA exit site of HMGB1 domain A were found to be essential for promoting RAG1/2-RSS complex formation. Because the phenylalanine wedge is required for specific recognition of DNA kinks, we propose that HMGB proteins facilitate RAG1/2-RSS interactions by recognizing a distorted DNA structure induced by RAG1/2 binding. The resulting complex must be sufficiently dynamic to enable the series of RAG1/2-mediated chemical reactions on the DNA.

---

* Corresponding author. Mailing address for Reid C. Johnson: Department of Biological Chemistry, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, CA 90095-1737. Phone: (310) 825-7800. Fax: (310) 206-5272. E-mail: rcjohnson{at}mednet.ucla.edu. Mailing address for Marjorie A. Oettinger: 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.

Present address: Cancer Research Center, Boston University School of Medicine, 715 Albany St., Boston, MA 02118.

Present address: Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02142.

Present address: Division of Molecular Life Sciences and Center for Cell Signaling Research, Ewha Womans University, Seoul 120-750, South Korea.

---

Molecular and Cellular Biology, June 2005, p. 4413-4425, Vol. 25, No. 11
0022-538X/05/$08.00+0     doi:10.1128/MCB.25.11.4413-4425.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

This article has been cited by other articles:

• Hystad, M. E., Myklebust, J. H., Bo, T. H., Sivertsen, E. A., Rian, E., Forfang, L., Munthe, E., Rosenwald, A., Chiorazzi, M., Jonassen, I., Staudt, L. M., Smeland, E. B. (2007). Characterization of Early Stages of Human B Cell Development by Gene Expression Profiling. J. Immunol. 179: 3662-3671 [Abstract] [Full Text]  
• Ciubotaru, M., Kriatchko, A. N., Swanson, P. C., Bright, F. V., Schatz, D. G. (2007). Fluorescence Resonance Energy Transfer Analysis of Recombination Signal Sequence Configuration in the RAG1/2 Synaptic Complex. Mol. Cell. Biol. 27: 4745-4758 [Abstract] [Full Text]