Identification of basic residues in RAG2 critical for DNA binding by the RAG1-RAG2 complex.
In V(D)J recombination, the RAG1 and RAG2 proteins are the essential components of the complex that catalyzes DNA cleavage. RAG1 has been shown to play a central role in DNA binding and catalysis. In contrast, the molecular roles of RAG2 in V(D)J recombination are unknown. To address this, we individually mutated 36 evolutionarily conserved basic and hydroxy group containing residues within RAG2. Biochemical analysis of the recombinant RAG2 proteins led to the identification of a number of basic residue mutants defective in catalysis in vitro and V(D)J recombination in vivo. Five of these were deficient in binding of the RAG1-RAG2 complex to its cognate DNA target sequence while interacting normally with RAG1. Our findings provide support for the direct involvement of RAG2 in DNA binding during all steps of the cleavage reaction.
Fugmann
S D
SD
Section of Immunobiology, Howard Hughes Medical Institute, Yale School of Medicine, New Haven, CT 06520, USA.
Schatz
D G
DG
D004247:DNA / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D017930:Genes, Reporter / Q000235:genetics; D018398:Homeodomain Proteins / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D009690:Nucleic Acid Conformation; D009841:Oligonucleotides / Q000378:metabolism; D017354:Point Mutation; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D011995:Recombination, Genetic; D020798:Two-Hybrid System Techniques
D004268:DNA-Binding Proteins; D018398:Homeodomain Proteins; D009841:Oligonucleotides; D011994:Recombinant Proteins; C064655:V(D)J recombination activating protein 2; C064658:RAG-1 protein; D004247:DNA