V(D)J recombination catalyzed by mutant RAG proteins lacking consensus DNA-PK phosphorylation sites

The process of antigen receptor gene rearrangement, V(D)J recombination, in volves DNA cleavage by the RAG-1 and RAG-2 proteins. Cleavage generates cov alently sealed (hairpin) DNA ends, termed coding ends, which must be opened by an endonuclease prior to joining. Resolution of these hairpin ends requ ires the activity of the DNA-dependent protein kinase (DNA-PK), a protein k inase whose specific role is yet undetermined. It has been suggested that p hosphorylation of one or both RAG proteins by DNA-PK is required to activat e or recruit the hairpin-opening nuclease. Furthermore, very recent work ha s shown that RAG proteins themselves can open hairpins. These data raise th e possibility that DNA-PK-mediated phosphorylation of the RAG proteins coul d regulate the hairpin opening reaction. To test this hypothesis, we constr ucted mutant versions of RAG-1 and RAG-2 in which all four DNA-PK consensus phosphorylation sites were removed by site-directed mutagenesis. Our data provide conclusive evidence that phosphorylation of these conserved serine/ threonine residues is not required for hairpin opening or joining of V(D)J recombination intermediates. (C) 2000 Elsevier Science Ltd. All rights rese rved.