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Originally published In Press as doi:10.1074/jbc.M209758200 on December 17, 2002

J. Biol. Chem., Vol. 278, Issue 8, 5584-5596, February 21, 2003
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RAG1-DNA Binding in V(D)J Recombination
SPECIFICITY AND DNA-INDUCED CONFORMATIONAL CHANGES REVEALED BY FLUORESCENCE AND CD SPECTROSCOPY*

Mihai CiubotaruDagger §, Leon M. Ptaszek, Gary A. Baker||, Sheila N. Baker||, Frank V. Bright||, and David G. SchatzDagger **

From the Dagger  Howard Hughes Medical Institute, Yale University School of Medicine, Section of Immunobiology, New Haven, Connecticut 06510, || State University of New York at Buffalo, Department of Chemistry, Amherst, New York 12640, and  Mount Sinai School of Medicine, New York University, New York, New York 10029

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 KD = 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.

* This work was supported by National Institutes of Health Grant AI32524 (to D. G. S.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ A postdoctoral associate.

** An investigator of the Howard Hughes Medical Institute. To whom correspondence should be addressed: Howard Hughes Medical Institute, Yale University School of Medicine, Section of Immunobiology, 310 Cedar St., New Haven, CT 06510. Tel.: 203-737-2255; Fax: 203-737-1764; E-mail: david.schatz@yale.edu.

Copyright © 2003 by The American Society for Biochemistry and Molecular Biology, Inc.
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