RAG and HMGB1 create a large bend in the 23RSS in the V(D)J recombination synaptic complexes

Department of Immunobiology, Yale University School of Medicine, 300 Cedar St., New Haven, CT 06511, USA, Department of Enzymology, Institute of Biochemistry of the Romanian Academy, Splaiul Independentei 296, 060031 Bucharest, Romania, Horia Hulubei National Institute for Physics and Nuclear Engineering, 077125 Bucharest-Magurele, Romania, Department of Molecular Biophysics and Biochemistry, Yale University School of Medicine, 300 Cedar St., New Haven, CT 06511, USA, Department of Bioinformatics and Structural Biochemistry, Institute of Biochemistry of the Romanian Academy, Splaiul Independentei 296, 060031 Bucharest, Romania and Howard Hughes Medical Institute, 4000 Jones Bridge Road Chevy Chase, MD 20815, USA.
Nucleic Acids Research (Impact Factor: 9.11). 01/2013; 41(4). DOI: 10.1093/nar/gks1294
Source: PubMed

ABSTRACT During V(D)J recombination, recombination activating gene proteins RAG1 and RAG2 generate DNA double strand breaks within a paired complex (PC) containing two complementary recombination signal sequences (RSSs), the 12RSS and 23RSS, which differ in the length of the spacer separating heptamer and nonamer elements. Despite the central role of the PC in V(D)J recombination, little is understood about its structure. Here, we use fluorescence resonance energy transfer to investigate the architecture of the 23RSS in the PC. Energy transfer was detected in 23RSS substrates in which the donor and acceptor fluorophores flanked the entire RSS, and was optimal under conditions that yield a cleavage-competent PC. The data are most easily explained by a dramatic bend in the 23RSS that reduces the distance between these flanking regions from >160 Å in the linear substrate to <80 Å in the PC. Analysis of multiple fluorescent substrates together with molecular dynamics modeling yielded a model in which the 23RSS adopts a U shape in the PC, with the spacer located centrally within the bend. We propose that this large bend facilitates simultaneous recognition of the heptamer and nonamer, is critical for proper positioning of the active site and contributes to the 12/23 rule.

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Available from: Marius Surleac, Mar 20, 2014
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    ABSTRACT: During V(D)J recombination, recombination activating gene (RAG)1 and RAG2 bind and cleave recombination signal sequences (RSSs), aided by the ubiquitous DNA-binding/-bending proteins high-mobility group box protein (HMGB)1 or HMGB2. HMGB1/2 play a critical, although poorly understood, role in vitro in the assembly of functional RAG-RSS complexes, into which HMGB1/2 stably incorporate. The mechanism of HMGB1/2 recruitment is unknown, although an interaction with RAG1 has been suggested. Here, we report data demonstrating only a weak HMGB1-RAG1 interaction in the absence of DNA in several assays, including fluorescence anisotropy experiments using a novel Alexa488-labeled HMGB1 protein. Addition of DNA to RAG1 and HMGB1 in fluorescence anisotropy experiments, however, results in a substantial increase in complex formation, indicating a synergistic binding effect. Pulldown experiments confirmed these results, as HMGB1 was recruited to a RAG1-DNA complex in a RAG1 concentration-dependent manner and, interestingly, without strict RSS sequence specificity. Our finding that HMGB1 binds more tightly to a RAG1-DNA complex over RAG1 or DNA alone provides an explanation for the stable integration of this typically transient architectural protein in the V(D)J recombinase complex throughout recombination. These findings also have implications for the order of events during RAG-DNA complex assembly and for the stabilization of sequence-specific and non-specific RAG1-DNA interactions.
    Nucleic Acids Research 01/2013; 41(5). DOI:10.1093/nar/gks1461 · 9.11 Impact Factor
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    ABSTRACT: At the Tcrb locus, Vβ-to-Jβ rearrangement is permitted by the 12/23 rule but is not observed in vivo, a restriction termed the Beyond 12/23 rule (B12/23 rule). Previous work showed that Vβ RSSs do not recombine with Jβ RSSs because Jβ RSSs are crippled for either nicking or synapsis. This result raised the question: how can crippled Jβ RSSs recombine with Dβ RSSs? We report here that the nicking of some Jβ RSSs can be substantially stimulated by synapsis with a 3'Dβ1 partner RSS. This result helps to reconcile disagreement in the field regarding the impact of synapsis on nicking. Furthermore, our data allow for the classification of Tcrb RSSs into two major categories: those that nick quickly and those that nick slowly in the absence of partner. Slow-nicking RSSs can be stimulated to nick more efficiently upon synapsis with an appropriate B12/23 partner, and our data unexpectedly suggest that fast-nicking RSSs can be inhibited for nicking upon synapsis with an inappropriate partner. These observations indicate that the RAG proteins exert fine control over every step of V(D)J cleavage and support the hypothesis that initial RAG binding can occur on either 12- or 23-RSSs.
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