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Molecular and Cellular Biology, October 2004, p. 8727-8744, Vol. 24, No. 19
0270-7306/04/$08.00+0     DOI: 10.1128/MCB.24.19.8727-8744.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Synapsis of Recombination Signal Sequences Located in cis and DNA Underwinding in V(D)J Recombination

Mihai Ciubotaru and David G. Schatz*

Section of Immunobiology, Howard Hughes Medical Institute, Yale University School of Medicine, New Haven, Connecticut

Received 2 December 2003/ Returned for modification 21 February 2004/ Accepted 22 June 2004

V(D)J recombination requires binding and synapsis of a complementary (12/23) pair of recombination signal sequences (RSSs) by the RAG1 and RAG2 proteins, aided by a high-mobility group protein, HMG1 or HMG2. Double-strand DNA cleavage within this synaptic, or paired, complex is thought to involve DNA distortion or melting near the site of cleavage. Although V(D)J recombination normally occurs between RSSs located on the same DNA molecule (in cis), all previous studies that directly assessed RSS synapsis were performed with the two DNA substrates in trans. To overcome this limitation, we have developed a facilitated circularization assay using DNA substrates of reduced length to assess synapsis of RSSs in cis. We show that a 12/23 pair of RSSs is the preferred substrate for synapsis of cis RSSs and that the efficiency of pairing is dependent upon RAG1-RAG2 stoichiometry. Synapsis in cis occurs rapidly and is kinetically favored over synapsis of RSSs located in trans. This experimental system also allowed the generation of underwound DNA substrates containing pairs of RSSs in cis. Importantly, we found that the RAG proteins cleave such substrates substantially more efficiently than relaxed substrates and that underwinding may enhance RSS synapsis as well as RAG1/2-mediated catalysis. The energy stored in such underwound substrates may be used in the generation of DNA distortion and/or protein conformational changes needed for synapsis and cleavage. We propose that this unwinding is uniquely sensed during synapsis of an appropriate 12/23 pair of RSSs.

* Corresponding author. Mailing address: Section of Immunobiology, Howard Hughes Medical Institute, Yale University School of Medicine, 300 Cedar St., TAC S625, New Haven, CT 06510. Phone: (203) 737-2255. Fax: (203) 737-1764. E-mail: david.schatz{at}yale.edu.

Molecular and Cellular Biology, October 2004, p. 8727-8744, Vol. 24, No. 19
0022-538X/04/$08.00+0     DOI: 10.1128/MCB.24.19.8727-8744.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

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