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Mol Cell Biol, August 1998, p. 4670-4678, Vol. 18, No. 8
0270-7306/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.

Distinct Roles of RAG1 and RAG2 in Binding the V(D)J Recombination Signal Sequences

Yoshiko Akamatsu and Marjorie A. Oettinger*

Department of Molecular Biology, Massachusetts General Hospital, Boston, Massachusetts 02114, and Department of Genetics, Harvard Medical School, Boston, Massachusetts 02115

Received 29 December 1997/Returned for modification 15 February 1998/Accepted 15 May 1998

The RAG1 and RAG2 proteins initiate V(D)J recombination by introducing double-strand breaks at the border between a recombination signal sequence (RSS) and a coding segment. To understand the distinct functions of RAG1 and RAG2 in signal recognition, we have compared the DNA binding activities of RAG1 alone and RAG1 plus RAG2 by gel retardation and footprinting analyses. RAG1 exhibits only a three- to fivefold preference for binding DNA containing an RSS over random sequence DNA. Although direct binding of RAG2 by itself was not detected, the presence of both RAG1 and RAG2 results in the formation of a RAG1-RAG2-DNA complex which is more stable and more specific than the RAG1-DNA complex and is active in V(D)J cleavage. These results suggest that biologically effective discrimination between an RSS and nonspecific sequences requires both RAG1 and RAG2. Unlike the binding of RAG1 plus RAG2, RAG1 can bind to DNA in the absence of a divalent metal ion and does not require the presence of coding flank sequence. Footprinting of the RAG1-RAG2 complex with 1,10-phenanthroline-copper and dimethyl sulfate protection reveal that both the heptamer and the nonamer are involved. The nonamer is protected, with extensive protein contacts within the minor groove. Conversely, the heptamer is rendered more accessible to chemical attack, suggesting that binding of RAG1 plus RAG2 distorts the DNA near the coding/signal border.

* Corresponding author. Mailing address: Department of Molecular Biology, Massachusetts General Hospital, Boston, MA 02115. Phone: (617) 726-5967. Fax: (617) 726-5949. E-mail: oettinger{at}frodo.mgh.harvard.edu.

Mol Cell Biol, August 1998, p. 4670-4678, Vol. 18, No. 8
0270-7306/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.


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