Detection of RAG protein-V(D)J recombination signal interactions near the site of DNA cleavage by UV cross-linking.
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.
Eastman
Q M
QM
Department of Molecular Biophysics and Biochemistry, Yale University School of Medicine, New Haven, Connecticut 06520-8011, USA.
Villey
I J
IJ
Schatz
D G
DG
D000818:Animals; D001665:Binding Sites / Q000235:genetics; D002460:Cell Line; D003432:Cross-Linking Reagents / Q000378:metabolism; D004247:DNA / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000235:genetics; D005803:Genes, Immunoglobulin / Q000235:genetics*; D018398:Homeodomain Proteins / Q000235:genetics*; D051379:Mice; D008958:Models, Molecular; D009154:Mutation / Q000235:genetics; D011995:Recombination, Genetic / Q000235:genetics*; D014466:Ultraviolet Rays
D003432:Cross-Linking Reagents; D004268:DNA-Binding Proteins; D018398:Homeodomain Proteins; C499308:Rag2 protein, mouse; C064655:V(D)J recombination activating protein 2; C064658:RAG-1 protein; D004247:DNA