title: Cooperative recruitment of HMGB1 during V(D)J recombination through interactions with RAG1 and DNA.
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.
authors: Department of Immunobiology, Yale University School of Medicine, 300 Cedar Street, New Haven, CT 06511, USA.
authors: Alicia J
authors: AJ
authors: Little
authors: Elizabeth
authors: E
authors: Corbett
authors: Fabian
authors: F
authors: Ortega
authors: David G
authors: DG
authors: Schatz
mesh_terms: D019943:Amino Acid Substitution; D000818:Animals; D002850:Chromatography, Gel; D004247:DNA / Q000737:chemistry* / Q000378:metabolism; D057809:HEK293 Cells; D024243:HMGB1 Protein / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D018398:Homeodomain Proteins / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D051379:Mice; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D012045:Regulatory Sequences, Nucleic Acid; D060152:V(D)J Recombination
chemical_list: D024243:HMGB1 Protein; D018398:Homeodomain Proteins; C064658:RAG-1 protein; D004247:DNA