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The RAG-HMG1 Complex Enforces the 12/23 Rule of V(D)J Recombination Specifically at the Double-Hairpin Formation Step

The RAG-HMG1 Complex Enforces the 12/23 Rule of V(D)J Recombination Specifically at the... The RAG-HMG1 Complex Enforces the 12/23 Rule of V(D)J Recombination Specifically at the Double-Hairpin Formation Step Robert B. West and Michael R. Lieber * Norris Comprehensive Cancer Center, Departments of Pathology, Biochemistry and Molecular Biology, and Molecular Microbiology and Immunology, University of Southern California School of Medicine, Los Angeles, California 90033 ABSTRACT A central unanswered question concerning the initial phases of V(D)J recombination has been at which step the 12/23 rule applies. This rule, which governs which variable (V), diversity (D), and joining (J) segments are able to pair during recombination, could operate at the level of signal sequence synapsis after RAG-HMG1 complex binding, signal nicking, or signal hairpin formation. It has also been unclear whether additional proteins are required to achieve adherence to the 12/23 rule. We developed a novel system for the detailed biochemical analysis of the 12/23 rule by using an oligonucleotide-based substrate that can include two signals. Under physiologic conditions, we found that the complex of RAG1, RAG2, and HMG1 can successfully recapitulate the 12/23 rule with the same specificity as that seen intracellularly and in crude extracts. The cleavage complex can bind and nick 12×12 and 23×23 substrates as well as 12×23 substrates. However, hairpin formation occurs at both of the signals only on 12×23 substrates. Moreover, under physiologic conditions, the presence of a partner 23-bp spacer suppresses single-site hairpin formation at a 12-bp spacer and vice versa. Hence, this study illustrates that synapsis suppresses single-site reactions, thereby explaining the high physiologic ratio of paired versus unpaired V(D)J recombination events in lymphoid cells. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Molecular and Cellular Biology American Society For Microbiology

The RAG-HMG1 Complex Enforces the 12/23 Rule of V(D)J Recombination Specifically at the Double-Hairpin Formation Step

Molecular and Cellular Biology , Volume 18 (11): 6408 – Nov 1, 1998

The RAG-HMG1 Complex Enforces the 12/23 Rule of V(D)J Recombination Specifically at the Double-Hairpin Formation Step

Molecular and Cellular Biology , Volume 18 (11): 6408 – Nov 1, 1998

Abstract

The RAG-HMG1 Complex Enforces the 12/23 Rule of V(D)J Recombination Specifically at the Double-Hairpin Formation Step Robert B. West and Michael R. Lieber * Norris Comprehensive Cancer Center, Departments of Pathology, Biochemistry and Molecular Biology, and Molecular Microbiology and Immunology, University of Southern California School of Medicine, Los Angeles, California 90033 ABSTRACT A central unanswered question concerning the initial phases of V(D)J recombination has been at which step the 12/23 rule applies. This rule, which governs which variable (V), diversity (D), and joining (J) segments are able to pair during recombination, could operate at the level of signal sequence synapsis after RAG-HMG1 complex binding, signal nicking, or signal hairpin formation. It has also been unclear whether additional proteins are required to achieve adherence to the 12/23 rule. We developed a novel system for the detailed biochemical analysis of the 12/23 rule by using an oligonucleotide-based substrate that can include two signals. Under physiologic conditions, we found that the complex of RAG1, RAG2, and HMG1 can successfully recapitulate the 12/23 rule with the same specificity as that seen intracellularly and in crude extracts. The cleavage complex can bind and nick 12×12 and 23×23 substrates as well as 12×23 substrates. However, hairpin formation occurs at both of the signals only on 12×23 substrates. Moreover, under physiologic conditions, the presence of a partner 23-bp spacer suppresses single-site hairpin formation at a 12-bp spacer and vice versa. Hence, this study illustrates that synapsis suppresses single-site reactions, thereby explaining the high physiologic ratio of paired versus unpaired V(D)J recombination events in lymphoid cells.

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Publisher
American Society For Microbiology
Copyright
Copyright © 1998 by the American society for Microbiology.
ISSN
0270-7306
eISSN
1098-5549
Publisher site
See Article on Publisher Site

Abstract

The RAG-HMG1 Complex Enforces the 12/23 Rule of V(D)J Recombination Specifically at the Double-Hairpin Formation Step Robert B. West and Michael R. Lieber * Norris Comprehensive Cancer Center, Departments of Pathology, Biochemistry and Molecular Biology, and Molecular Microbiology and Immunology, University of Southern California School of Medicine, Los Angeles, California 90033 ABSTRACT A central unanswered question concerning the initial phases of V(D)J recombination has been at which step the 12/23 rule applies. This rule, which governs which variable (V), diversity (D), and joining (J) segments are able to pair during recombination, could operate at the level of signal sequence synapsis after RAG-HMG1 complex binding, signal nicking, or signal hairpin formation. It has also been unclear whether additional proteins are required to achieve adherence to the 12/23 rule. We developed a novel system for the detailed biochemical analysis of the 12/23 rule by using an oligonucleotide-based substrate that can include two signals. Under physiologic conditions, we found that the complex of RAG1, RAG2, and HMG1 can successfully recapitulate the 12/23 rule with the same specificity as that seen intracellularly and in crude extracts. The cleavage complex can bind and nick 12×12 and 23×23 substrates as well as 12×23 substrates. However, hairpin formation occurs at both of the signals only on 12×23 substrates. Moreover, under physiologic conditions, the presence of a partner 23-bp spacer suppresses single-site hairpin formation at a 12-bp spacer and vice versa. Hence, this study illustrates that synapsis suppresses single-site reactions, thereby explaining the high physiologic ratio of paired versus unpaired V(D)J recombination events in lymphoid cells.

Journal

Molecular and Cellular BiologyAmerican Society For Microbiology

Published: Nov 1, 1998

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