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Molecular and Cellular Biology, November 2009, p. 5889-5899, Vol. 29, No. 21
0270-7306/09/$08.00+0     doi:10.1128/MCB.00187-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Base Flipping in V(D)J Recombination: Insights into the Mechanism of Hairpin Formation, the 12/23 Rule, and the Coordination of Double-Strand Breaks ^,

Julien Bischerour,^1, Catherine Lu,² David B. Roth,² and Ronald Chalmers^1*

University of Nottingham, School of Biomedical Sciences, Medical School, Queen's Medical Centre, Nottingham NG7 2UH, United Kingdom,¹ Kimmel Center for Biology and Medicine of the Skirball Institute and Department of Pathology, New York University School of Medicine, New York, New York 10016²

Received 10 February 2009/ Returned for modification 3 March 2009/ Accepted 21 August 2009

Tn5 transposase cleaves the transposon end using a hairpin intermediate on the transposon end. This involves a flipped base that is stacked against a tryptophan residue in the protein. However, many other members of the cut-and-paste transposase family, including the RAG1 protein, produce a hairpin on the flanking DNA. We have investigated the reversed polarity of the reaction for RAG recombination. Although the RAG proteins appear to employ a base-flipping mechanism using aromatic residues, the putatively flipped base is not at the expected location and does not appear to stack against any of the said aromatic residues. We propose an alternative model in which a flipped base is accommodated in a nonspecific pocket or cleft within the recombinase. This is consistent with the location of the flipped base at position –1 in the coding flank, which can be occupied by purine or pyrimidine bases that would be difficult to stabilize using a single, highly specific, interaction. Finally, during this work we noticed that the putative base-flipping events on either side of the 12/23 recombination signal sequence paired complex are coupled to the nicking steps and serve to coordinate the double-strand breaks on either side of the complex.

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* Corresponding author. Mailing address: University of Nottingham, School of Biomedical Sciences, Medical School, Queen's Medical Centre, Nottingham NG7 2UH, United Kingdom. Phone: 44 115 823 0087. Fax: 44 115 823 0103. E-mail: chalmers{at}nottingham.ac.uk

Published ahead of print on 31 August 2009.

Supplemental material for this article may be found at http://mcb.asm.org/.

Present address: Centre de Genetique Moleculaire, CNRS UPR2167, Avenue de la Terrasse, 91198 Gif sur Yvette Cedex, France.

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Molecular and Cellular Biology, November 2009, p. 5889-5899, Vol. 29, No. 21
0270-7306/09/$08.00+0     doi:10.1128/MCB.00187-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

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