RAG1-DNA binding in V(D)J recombination. Specificity and DNA-induced conformational changes revealed by fluorescence and CD spectroscopy.
The RAG1 and RAG2 proteins together constitute the nuclease that initiates the assembly of immunoglobulin and T cell receptor genes in a reaction known as V(D)J recombination. RAG1 plays a central role in recognition of the recombination signal sequence (RSS) by the RAG1/2 complex. To investigate the parameters governing the RAG1-RSS interaction, the murine core RAG1 protein (amino acids 377-1008) fused to a short Strep tag has been purified to homogeneity from bacteria. The Strep-RAG1 (StrRAG1) protein exists as a dimer at a wide range of protein concentrations (25-500 nM) in the absence of DNA and binds with reasonably high affinity and specificity (apparent K(D) = 41 nM) to the RSS. Both electrophoretic mobility shift assays and polarization anisotropy experiments indicate that only a single StrRAG1-DNA species exists in solution. Anisotropy decay measured by frequency domain spectroscopy suggests that the complex contains a dimer of StrRAG1 bound to a single DNA molecule. Using measurements of protein intrinsic fluorescence and circular dichroism, we demonstrate that StrRAG1 undergoes a major conformational change upon binding the RSS. Steady-state fluorescence and acrylamide quenching studies reveal that this conformational change is associated with a repositioning of intrinsic protein fluorophores from a hydrophobic to a solvent-exposed environment. RSS-induced conformational changes of StrRAG1 may influence the interaction of RAG1 with RAG2 and synaptic complex formation.
Ciubotaru
Mihai
M
Howard Hughes Medical Institute, Yale University School of Medicine, Section of Immunobiology, New Haven, Connecticut 06510, USA.
Ptaszek
Leon M
LM
Baker
Gary A
GA
Baker
Sheila N
SN
Bright
Frank V
FV
Schatz
David G
DG
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002942:Circular Dichroism; D003001:Cloning, Molecular; D004247:DNA / Q000737:chemistry / Q000378:metabolism*; D004254:DNA Nucleotidyltransferases / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D016654:Genes, RAG-1; D018398:Homeodomain Proteins / Q000737:chemistry* / Q000378:metabolism*; D007700:Kinetics; D051379:Mice; D009838:Oligodeoxyribonucleotides / Q000378:metabolism; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000378:metabolism; D011995:Recombination, Genetic; D013379:Substrate Specificity; D014162:Transfection; D019895:Transposases / Q000737:chemistry / Q000378:metabolism; D045567:VDJ Recombinases
D004268:DNA-Binding Proteins; D018398:Homeodomain Proteins; D009838:Oligodeoxyribonucleotides; C499308:Rag2 protein, mouse; D011994:Recombinant Proteins; C064655:V(D)J recombination activating protein 2; C064658:RAG-1 protein; D004247:DNA; D004254:DNA Nucleotidyltransferases; D019895:Transposases; D045567:VDJ Recombinases