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Molecular and Cellular Biology, May 1999, p. 3788-3797, Vol. 19, No. 5
0270-7306/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.

Detection of RAG Protein-V(D)J Recombination Signal Interactions Near the Site of DNA Cleavage by UV Cross-Linking

Quinn M. Eastman,1 Isabelle J. Villey,2,3 and David G. Schatz2,3,*

Department of Molecular Biophysics and Biochemistry,1 Howard Hughes Medical Institute,2 and Section of Immunobiology,3 Yale University School of Medicine, New Haven, Connecticut 06520-8011

Received 18 September 1998/Returned for modification 2 November 1998/Accepted 27 January 1999

V(D)J recombination is initiated by double-strand cleavage at recombination signal sequences (RSSs). DNA cleavage is mediated by the RAG1 and RAG2 proteins. Recent experiments describing RAG protein-RSS complexes, while defining the interaction of RAG1 with the nonamer, have not assigned contacts immediately adjacent to the site of DNA cleavage to either RAG polypeptide. Here we use UV cross-linking to define sequence- and site-specific interactions between RAG1 protein and both the heptamer element of the RSS and the coding flank DNA. Hence, RAG1-DNA contacts span the site of cleavage. We also detect cross-linking of RAG2 protein to some of the same nucleotides that cross-link to RAG1, indicating that, in the binding complex, both RAG proteins are in close proximity to the site of cleavage. These results suggest how the heptamer element, the recognition surface essential for DNA cleavage, is recognized by the RAG proteins and have implications for the stoichiometry and active site organization of the RAG1-RAG2-RSS complex.

* Corresponding author. Mailing address: Section of Immunobiology, Yale University School of Medicine, 310 Cedar St., P.O. Box 208011, New Haven, CT 06520-8011. Phone: (203) 737-2255. Fax: (203) 737-1764. E-mail: david.schatz{at}yale.edu.

Molecular and Cellular Biology, May 1999, p. 3788-3797, Vol. 19, No. 5
0270-7306/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.


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