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A functional analysis of the spacer of V(D)J recombination signal sequences.

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Date
2003-10
Authors
Lee, Alfred Ian
Fugmann, Sebastian D
Cowell, Lindsay G
Ptaszek, Leon M
Kelsoe, Garnett
Schatz, David G
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Abstract
During lymphocyte development, V(D)J recombination assembles antigen receptor genes from component V, D, and J gene segments. These gene segments are flanked by a recombination signal sequence (RSS), which serves as the binding site for the recombination machinery. The murine Jbeta2.6 gene segment is a recombinationally inactive pseudogene, but examination of its RSS reveals no obvious reason for its failure to recombine. Mutagenesis of the Jbeta2.6 RSS demonstrates that the sequences of the heptamer, nonamer, and spacer are all important. Strikingly, changes solely in the spacer sequence can result in dramatic differences in the level of recombination. The subsequent analysis of a library of more than 4,000 spacer variants revealed that spacer residues of particular functional importance are correlated with their degree of conservation. Biochemical assays indicate distinct cooperation between the spacer and heptamer/nonamer along each step of the reaction pathway. The results suggest that the spacer serves not only to ensure the appropriate distance between the heptamer and nonamer but also regulates RSS activity by providing additional RAG:RSS interaction surfaces. We conclude that while RSSs are defined by a "digital" requirement for absolutely conserved nucleotides, the quality of RSS function is determined in an "analog" manner by numerous complex interactions between the RAG proteins and the less-well conserved nucleotides in the heptamer, the nonamer, and, importantly, the spacer. Those modulatory effects are accurately predicted by a new computational algorithm for "RSS information content." The interplay between such binary and multiplicative modes of interactions provides a general model for analyzing protein-DNA interactions in various biological systems.
Type
Journal article
Subject
Algorithms
Animals
Cell Line
Cloning, Molecular
Computational Biology
DNA
DNA, Intergenic
Humans
Lymphocytes
Mice
Models, Genetic
Models, Statistical
Mutagenesis
Oligonucleotides
Plasmids
Protein Binding
Protein Sorting Signals
Recombination, Genetic
Software
T-Lymphocytes
VDJ Recombinases
Permalink
https://hdl.handle.net/10161/11485
Published Version (Please cite this version)
10.1371/journal.pbio.0000001
Publication Info
Lee, Alfred Ian; Fugmann, Sebastian D; Cowell, Lindsay G; Ptaszek, Leon M; Kelsoe, Garnett; & Schatz, David G (2003). A functional analysis of the spacer of V(D)J recombination signal sequences. PLoS Biol, 1(1). pp. E1. 10.1371/journal.pbio.0000001. Retrieved from https://hdl.handle.net/10161/11485.
This is constructed from limited available data and may be imprecise. To cite this article, please review & use the official citation provided by the journal.
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Scholars@Duke

Cowell

Lindsay Grey Cowell

Adjunct Assistant Professor in the Department of Biostatistics and Bioinformatics
Somatic Diversification of Lymphocyte Antigen Receptor Genes * V(D)J Recombination * Somatic Hypermutation Biomedical Ontology * Ontological Representation of Cells of Hematopoietic Lineage Biomedical Text Mining Logic-based Reasoning
Kelsoe

Garnett H. Kelsoe

James B. Duke Distinguished Professor of Immunology
1. Lymphocyte development and antigen-driven diversification of immunoglobulin and T cell antigen receptor genes. 2. The germinal center reaction and mechanisms for clonal selection and self - tolerance. The origins of autoimmunity. 3. Interaction of innate- and adaptive immunity and the role of inflammation in lymphoid organogenesis. 4. The role of secondary V(D)J gene rearrangment in lymphocyte development and malignancies. 5. Mathematical modeling of immune responses,
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