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Molecular and Cellular Biology, October 1999, p. 6532-6542, Vol. 19, No. 10
0270-7306/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.

The RAG1 Homeodomain Recruits HMG1 and HMG2 To Facilitate Recombination Signal Sequence Binding and To Enhance the Intrinsic DNA-Bending Activity of RAG1-RAG2

Vassilis Aidinis,1,dagger Tiziana Bonaldi,2,3 Monica Beltrame,2 Sandro Santagata,1 Marco E. Bianchi,2,3,* and Eugenia Spanopoulou1,Dagger

Howard Hughes Medical Institute, Ruttenberg Cancer Center, Mount Sinai School of Medicine, New York, New York 10029,1 and Dipartimento di Genetica e di Biologia dei Microrganismi, 20133 Milan,2 and DIBIT, San Raffaele Scientific Institute, 20132 Milan,3 Italy

Received 10 February 1999/Returned for modification 12 March 1999/Accepted 28 June 1999

V(D)J recombination is initiated by the specific binding of the RAG1-RAG2 (RAG1/2) complex to the heptamer-nonamer recombination signal sequences (RSS). Several steps of the V(D)J recombination reaction can be reconstituted in vitro with only RAG1/2 plus the high-mobility-group protein HMG1 or HMG2. Here we show that the RAG1 homeodomain directly interacts with both HMG boxes of HMG1 and HMG2 (HMG1,2). This interaction facilitates the binding of RAG1/2 to the RSS, mainly by promoting high-affinity binding to the nonamer motif. Using circular-permutation assays, we found that the RAG1/2 complex bends the RSS DNA between the heptamer and nonamer motifs. HMG1,2 significantly enhance the binding and bending of the 23RSS but are not essential for the formation of a bent DNA intermediate on the 12RSS. A transient increase of HMG1,2 concentration in transfected cells increases the production of the final V(D)J recombinants in vivo.

* Corresponding author. Mailing address: Dipartimento di Genetica e di Biologia dei Microrganismi, via Celoria 26, 20133 Milan, Italy. Phone: 39-02.26.43.47.80. Fax: 39-02.26.43.48.61. E-mail: bianchi.marco{at}hsr.it.

dagger Present address: Laboratory of Molecular Genetics, Hellenic Pasteur Institute, 11521 Athens, Greece.

Dagger Eugenia Spanopoulou perished on 2 September 1998 in the crash of a Swissair flight between New York and Geneva.

Molecular and Cellular Biology, October 1999, p. 6532-6542, Vol. 19, No. 10
0270-7306/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.


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