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HMG-box domain stimulation of RAG1/2 cleavage activity is metal ion dependent

BACKGROUND: RAG1 and RAG2 initiate V(D)J recombination by assembling a synaptic complex with a pair of antigen receptor gene segments through interactions with their flanking recombination signal sequence (RSS), and then introducing a DNA double-strand break at each RSS, separating it from the adjac...

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Autores principales: Kriatchko, Aleksei N, Bergeron, Serge, Swanson, Patrick C
Formato: Texto
Lenguaje:English
Publicado: BioMed Central 2008
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2324110/
https://www.ncbi.nlm.nih.gov/pubmed/18380906
http://dx.doi.org/10.1186/1471-2199-9-32
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author Kriatchko, Aleksei N
Bergeron, Serge
Swanson, Patrick C
author_facet Kriatchko, Aleksei N
Bergeron, Serge
Swanson, Patrick C
author_sort Kriatchko, Aleksei N
collection PubMed
description BACKGROUND: RAG1 and RAG2 initiate V(D)J recombination by assembling a synaptic complex with a pair of antigen receptor gene segments through interactions with their flanking recombination signal sequence (RSS), and then introducing a DNA double-strand break at each RSS, separating it from the adjacent coding segment. While the RAG proteins are sufficient to mediate RSS binding and cleavage in vitro, these activities are stimulated by the architectural DNA binding and bending factors HMGB1 and HMGB2. Two previous studies (Bergeron et al., 2005, and Dai et al., 2005) came to different conclusions regarding whether only one of the two DNA binding domains of HMGB1 is sufficient to stimulate RAG-mediated binding and cleavage of naked DNA in vitro. Here we test whether this apparent discrepancy is attributed to the choice of divalent metal ion and the concentration of HMGB1 used in the cleavage reaction. RESULTS: We show here that single HMG-box domains of HMGB1 stimulate RAG-mediated RSS cleavage in a concentration-dependent manner in the presence of Mn(2+), but not Mg(2+). Interestingly, the inability of a single HMG-box domain to stimulate RAG-mediated RSS cleavage in Mg(2+ )is overcome by the addition of partner RSS to promote synapsis. Furthermore, we show that mutant forms of HMGB1 which otherwise fail to stimulate RAG-mediated RSS cleavage in Mg(2+ )can be substantially rescued when Mg(2+ )is replaced with Mn(2+). CONCLUSION: The conflicting data published previously in two different laboratories can be substantially explained by the choice of divalent metal ion and abundance of HMGB1 in the cleavage reaction. The observation that single HMG-box domains can promote RAG-mediated 23-RSS cleavage in Mg(2+ )in the presence, but not absence, of partner RSS suggests that synaptic complex assembly in vitro is associated with conformational changes that alter how the RAG and/or HMGB1 proteins bind and bend DNA in a manner that functionally replaces the role of one of the HMG-box domains in RAG-HMGB1 complexes assembled on a single RSS.
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spelling pubmed-23241102008-04-22 HMG-box domain stimulation of RAG1/2 cleavage activity is metal ion dependent Kriatchko, Aleksei N Bergeron, Serge Swanson, Patrick C BMC Mol Biol Research Article BACKGROUND: RAG1 and RAG2 initiate V(D)J recombination by assembling a synaptic complex with a pair of antigen receptor gene segments through interactions with their flanking recombination signal sequence (RSS), and then introducing a DNA double-strand break at each RSS, separating it from the adjacent coding segment. While the RAG proteins are sufficient to mediate RSS binding and cleavage in vitro, these activities are stimulated by the architectural DNA binding and bending factors HMGB1 and HMGB2. Two previous studies (Bergeron et al., 2005, and Dai et al., 2005) came to different conclusions regarding whether only one of the two DNA binding domains of HMGB1 is sufficient to stimulate RAG-mediated binding and cleavage of naked DNA in vitro. Here we test whether this apparent discrepancy is attributed to the choice of divalent metal ion and the concentration of HMGB1 used in the cleavage reaction. RESULTS: We show here that single HMG-box domains of HMGB1 stimulate RAG-mediated RSS cleavage in a concentration-dependent manner in the presence of Mn(2+), but not Mg(2+). Interestingly, the inability of a single HMG-box domain to stimulate RAG-mediated RSS cleavage in Mg(2+ )is overcome by the addition of partner RSS to promote synapsis. Furthermore, we show that mutant forms of HMGB1 which otherwise fail to stimulate RAG-mediated RSS cleavage in Mg(2+ )can be substantially rescued when Mg(2+ )is replaced with Mn(2+). CONCLUSION: The conflicting data published previously in two different laboratories can be substantially explained by the choice of divalent metal ion and abundance of HMGB1 in the cleavage reaction. The observation that single HMG-box domains can promote RAG-mediated 23-RSS cleavage in Mg(2+ )in the presence, but not absence, of partner RSS suggests that synaptic complex assembly in vitro is associated with conformational changes that alter how the RAG and/or HMGB1 proteins bind and bend DNA in a manner that functionally replaces the role of one of the HMG-box domains in RAG-HMGB1 complexes assembled on a single RSS. BioMed Central 2008-04-01 /pmc/articles/PMC2324110/ /pubmed/18380906 http://dx.doi.org/10.1186/1471-2199-9-32 Text en Copyright © 2008 Kriatchko et al; licensee BioMed Central Ltd. http://creativecommons.org/licenses/by/2.0 This is an Open Access article distributed under the terms of the Creative Commons Attribution License ( (http://creativecommons.org/licenses/by/2.0) ), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
spellingShingle Research Article
Kriatchko, Aleksei N
Bergeron, Serge
Swanson, Patrick C
HMG-box domain stimulation of RAG1/2 cleavage activity is metal ion dependent
title HMG-box domain stimulation of RAG1/2 cleavage activity is metal ion dependent
title_full HMG-box domain stimulation of RAG1/2 cleavage activity is metal ion dependent
title_fullStr HMG-box domain stimulation of RAG1/2 cleavage activity is metal ion dependent
title_full_unstemmed HMG-box domain stimulation of RAG1/2 cleavage activity is metal ion dependent
title_short HMG-box domain stimulation of RAG1/2 cleavage activity is metal ion dependent
title_sort hmg-box domain stimulation of rag1/2 cleavage activity is metal ion dependent
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2324110/
https://www.ncbi.nlm.nih.gov/pubmed/18380906
http://dx.doi.org/10.1186/1471-2199-9-32
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