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

Abstract

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.

ACKNOWLEDGMENTS

We thank Chia-Lun Tsai for assistance with RAG protein purification, Karla Rodgers for bacterial RAG1 protein and instruction on NAMOT and MOLMOL, the Schatz lab for patience with dimmed lights, Tad Koch for initial advice on cross-linking, and Eugenia Spanopoulou and Sankar Ghosh for helpful comments on the manuscript. We thank the W. M. Keck Foundation Biotechnology Resource Laboratory at Yale University for rapid oligonucleotide synthesis and the National Cell Culture Center (Minneapolis, Minn.) for large-scale F2A1 culture.

Q.M.E. was supported by a predoctoral fellowship from the National Science Foundation. I.J.V. was supported by an INSERM postdoctoral fellowship. This work was supported by Public Health Service grant AI-32524 from the National Institutes of Health. D.G.S. is an associate investigator of the Howard Hughes Medical Institute.