Nucleic Acids Research Advance Access originally published online on March 29, 2008
Nucleic Acids Research 2008 36(9):2864-2873; doi:10.1093/nar/gkn128
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Nucleic Acids Research, 2008, Vol. 36, No. 9 2864-2873
© 2008 The Author(s)
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Nucleic Acid Enzymes |
Understanding how the V(D)J recombinase catalyzes transesterification: distinctions between DNA cleavage and transposition
1Program in Molecular Pathogenesis, Helen L. and Martin S. Kimmel Center for Biology and Medicine at the Skirball Institute for Biomolecular Medicine, 2Department of Pathology, New York University School of Medicine, 540 First Avenue, New York, NY 10016 and 3Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX 77030, USA
*To whom correspondence should be addressed. Tel: +212 263 0945; Fax: +212 263 5711; Email: roth{at}saturn.med.nyu.edu
Received January 28, 2008. Revised February 29, 2008. Accepted March 5, 2008.
The Rag1 and Rag2 proteins initiate V(D)J recombination by introducing site-specific DNA double-strand breaks. Cleavage occurs by nicking one DNA strand, followed by a one-step transesterification reaction that forms a DNA hairpin structure. A similar reaction allows Rag transposition, in which the 3'-OH groups produced by Rag cleavage are joined to target DNA. The Rag1 active site DDE triad clearly plays a catalytic role in both cleavage and transposition, but no other residues in Rag1 responsible for transesterification have been identified. Furthermore, although Rag2 is essential for both cleavage and transposition, the nature of its involvement is unknown. Here, we identify basic amino acids in the catalytic core of Rag1 specifically important for transesterification. We also show that some Rag1 mutants with severe defects in hairpin formation nonetheless catalyze substantial levels of transposition. Lastly, we show that a catalytically defective Rag2 mutant is impaired in target capture and displays a novel form of coding flank sensitivity. These findings provide the first identification of components of Rag1 that are specifically required for transesterification and suggest an unexpected role for Rag2 in DNA cleavage and transposition.
The authors wish it to be known that, in their opinion, the first two authors should be regarded as joint First Authors
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