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

Published

Journal Article

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.

Full Text

Duke Authors

Cited Authors

  • Lee, AI; Fugmann, SD; Cowell, LG; Ptaszek, LM; Kelsoe, G; Schatz, DG

Published Date

  • October 1, 2003

Published In

Volume / Issue

  • 1 / 1

Start / End Page

  • E1 -

PubMed ID

  • 14551903

Pubmed Central ID

  • 14551903

Electronic International Standard Serial Number (EISSN)

  • 1545-7885

Digital Object Identifier (DOI)

  • 10.1371/journal.pbio.0000001

Language

  • eng

Conference Location

  • United States