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Molecular and Cellular Biology, June 1999, p. 4159-4166, Vol. 19, No. 6
0270-7306/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.

DNA Hairpin Opening Mediated by the RAG1 and RAG2 Proteins

Penny E. Shockett and David G. Schatz*

Howard Hughes Medical Institute and Section of Immunobiology, Yale University School of Medicine, New Haven, Connecticut 06520-8011

Received 14 January 1999/Returned for modification 19 February 1999/Accepted 15 March 1999

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.

* Corresponding author. Mailing address: Howard Hughes Medical Institute and 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 or (203) 737-1765. E-mail: david.schatz{at}yale.edu.

Molecular and Cellular Biology, June 1999, p. 4159-4166, Vol. 19, No. 6
0270-7306/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.


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