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Abstract
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.
Highlights
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
Three purification steps result in a StrRAG1 protein of greater than 98% purity (Fig. 1b)
When the purified dimeric protein was reanalyzed on the gel filtration column, only the dimer peak was observed, indicating that the protein remains stably associated in dimeric form (Fig. 2b)
Summary
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. Since most DNA binding studies have been performed by EMSA, we do not know the equilibrium constants governing reactions involving RAG1 alone or RAG1 in conjunction with RAG2 and HMG proteins in forming complexes with the RSS in solution.
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