The RAG1 homeodomain recruits HMG1 and HMG2 to facilitate recombination signal sequence binding and to enhance the intrinsic DNA-bending activity of RAG1-RAG2.
V(D)J recombination is initiated by the specific binding of the RAG1-RAG2 (RAG1/2) complex to the heptamer-nonamer recombination signal sequences (RSS). Several steps of the V(D)J recombination reaction can be reconstituted in vitro with only RAG1/2 plus the high-mobility-group protein HMG1 or HMG2. Here we show that the RAG1 homeodomain directly interacts with both HMG boxes of HMG1 and HMG2 (HMG1,2). This interaction facilitates the binding of RAG1/2 to the RSS, mainly by promoting high-affinity binding to the nonamer motif. Using circular-permutation assays, we found that the RAG1/2 complex bends the RSS DNA between the heptamer and nonamer motifs. HMG1,2 significantly enhance the binding and bending of the 23RSS but are not essential for the formation of a bent DNA intermediate on the 12RSS. A transient increase of HMG1,2 concentration in transfected cells increases the production of the final V(D)J recombinants in vivo.
Aidinis
V
V
Howard Hughes Medical Institute, Ruttenberg Cancer Center, Mount Sinai School of Medicine, New York, NY 10029, USA.
Bonaldi
T
T
Beltrame
M
M
Santagata
S
S
Bianchi
M E
ME
Spanopoulou
E
E
D001665:Binding Sites; D004268:DNA-Binding Proteins / Q000378:metabolism; D006609:High Mobility Group Proteins / Q000378:metabolism*; D018398:Homeodomain Proteins / Q000378:metabolism*; D009690:Nucleic Acid Conformation; D011485:Protein Binding; D011946:Receptors, Antigen / Q000235:genetics*; D011995:Recombination, Genetic
D004268:DNA-Binding Proteins; D006609:High Mobility Group Proteins; D018398:Homeodomain Proteins; D011946:Receptors, Antigen; C064655:V(D)J recombination activating protein 2; C064658:RAG-1 protein