Both high mobility group (HMG)-boxes and the acidic tail of HMGB1 regulate recombination-activating gene (RAG)-mediated recombination signal synapsis and cleavage in vitro.
RAG-1 and RAG-2 initiate V(D)J recombination through synapsis and cleavage of a 12/23 pair of V(D)J recombination signal sequences (RSS). RAG-RSS complex assembly and activity in vitro is promoted by high mobility group proteins of the "HMG-box" family, exemplified by HMGB1. How HMGB1 stimulates the DNA binding and cleavage activity of the RAG complex remains unclear. HMGB1 contains two homologous HMG-box DNA binding domains, termed A and B, linked by a stretch of basic residues to a highly acidic C-terminal tail. To identify determinants of HMGB1 required for stimulation of RAG-mediated RSS binding and cleavage, we prepared an extensive panel of mutant HMGB1 proteins and tested their ability to augment RAG-mediated RSS binding and cleavage activity. Using a combination of mobility shift and in-gel cleavage assays, we find that HMGB1 promotes RAG-mediated cleavage largely through the activity of box B, but optimal stimulation requires a functional A box tethered in the correct orientation. Box A or B mutants fail to promote RAG synaptic complex formation, but this defect is alleviated when the acidic tail is removed from these mutants.
Bergeron
Serge
S
Department of Medical Microbiology and Immunology, Creighton University Medical Center, Omaha, Nebraska 68178, USA.
Madathiparambil
Tina
T
Swanson
Patrick C
PC
D000818:Animals; D002460:Cell Line; D023902:Chromosome Pairing; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D024742:HMG-Box Domains; D024243:HMGB1 Protein / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D018398:Homeodomain Proteins / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D009687:Nuclear Proteins; D011485:Protein Binding; D020033:Protein Isoforms / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D011995:Recombination, Genetic
D004268:DNA-Binding Proteins; D024243:HMGB1 Protein; D018398:Homeodomain Proteins; D009687:Nuclear Proteins; D020033:Protein Isoforms; C499311:RAG2 protein, human; D011993:Recombinant Fusion Proteins; C064655:V(D)J recombination activating protein 2; C064658:RAG-1 protein