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Molecular and Cellular Biology, December 1999, p. 8094-8102, Vol. 19, No. 12
Norris Comprehensive Cancer Center,
Departments of Pathology, Biochemistry and Molecular Biology, and
Molecular Microbiology and Immunology, University of Southern
California School of Medicine, Los Angeles, California 90033
Received 14 July 1999/Returned for modification 16 August
1999/Accepted 13 September 1999
V(D)J recombination is directed by recombination signal sequences.
However, the flanking coding end sequence can markedly affect the
frequency of the initiation of V(D)J recombination in vivo. Here we
demonstrate that the coding end sequence effect can be qualitatively
and quantitatively recapitulated in vitro with purified RAG proteins.
We find that coding end sequence specifically affects the nicking step,
which is the first biochemical step in RAG-mediated cleavage. The
subsequent hairpin formation step is not affected by the coding end
sequence. Furthermore, the coding end sequence effect can be ablated by
prenicking the substrate, indicating that the coding end effect is
specific to the nicking step. In reactions in which both 12- and
23-substrates are present, a suboptimal coding end sequence on one
signal can slow down hairpin formation at the partner signal, a result
consistent with models in which coordination between the signals occurs
at the hairpin formation step. The coding end sequence effect on
nicking and the coupling of the 12- and 23-substrates explains how
hairpin formation can be rate limiting for some 12/23 pairs, whereas
nicking can be rate limiting when low-efficiency coding end sequences are involved.
0270-7306/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Mechanistic Basis for Coding End Sequence Effects
in the Initiation of V(D)J Recombination
*
Corresponding author. Norris Comprehensive Cancer
Center, Rm. 5428, Departments of Pathology, Biochemistry and Molecular
Biology, and Molecular Biology and Immunology, University of Southern
California School of Medicine, 1441 Eastlake Ave., Mail Stop 73, Los
Angeles, CA 90033. Phone: (323) 865-0568. Fax: (323) 865-3019. E-mail: lieber{at}hsc.usc.edu.
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