DNA hairpin opening mediated by the RAG1 and RAG2 proteins.
The lymphoid cell-specific proteins RAG1 and RAG2 initiate V(D)J recombination by cleaving DNA adjacent to recombination signals, generating blunt signal ends and covalently sealed, hairpin coding ends. A critical next step in the reaction is opening of the hairpins, but the factor(s) responsible has not been identified and had been thought to be a ubiquitous component(s) of the DNA repair machinery. Here we demonstrate that RAG1 and RAG2 possess an intrinsic single-stranded nuclease activity capable of nicking hairpin coding ends at or near the hairpin tip. In Mn2+, a synthetic hairpin is nicked 5 nucleotides (nt) 5' of the hairpin tip, with more distant sites of nicking suppressed by HMG2. In Mg2+, hairpins generated by V(D)J cleavage are nicked whereas synthetic hairpins are not. Cleavage-generated hairpins are nicked at the tip and predominantly 1 to 2 nt 5' of the tip. RAG1 and RAG2 may therefore be responsible for initiating the processing of coding ends and for the generation of P nucleotides during V(D)J recombination.
D015139:Blotting, Southern; D003001:Cloning, Molecular; D017931:DNA Primers; D004260:DNA Repair / Q000502:physiology*; D004268:DNA-Binding Proteins; D005092:Exonucleases / Q000378:metabolism; D018398:Homeodomain Proteins / Q000235:genetics*; D008274:Magnesium / Q000378:metabolism; D008345:Manganese / Q000378:metabolism; D008957:Models, Genetic; D016133:Polymerase Chain Reaction; D011995:Recombination, Genetic; D013997:Time Factors