A Functional Analysis of the Spacer of V(D)J Recombination Signal Sequences

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 Jβ2.6 gene segment is a recombinationally inactive pseudogene, but examination of its RSS reveals no obvious reason for its failure to recombine. Mutagenesis of the Jβ2.6 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.

V(D)J Recombinase Binding and Cleavage of Cryptic Recombination Signal Sequences Identified from Lymphoid Malignancies

“…2), as others have argued (31). We also acknowledge the possibility that distinct cRSSs may be similarly bound and cleaved by the RAG complex but support joining at different levels due to sequence variations (32).…”

Unifying model for molecular determinants of the preselection Vβ repertoire

“…As noted above, TRBV13-2 is also the most frequently rearranged gene segment in DN3 thymocytes, suggesting a dominant correlation between open chromatin and longrange recombination efficiency. Consistent with this possibility, many of the pseudogene segments, even those containing functional RSSs, are expressed at a low level and are associated with (40). Rearrangements were detected by PCR using primers shared by all of the substrates (NR, not rearranged; R, Vβ rearranged to Dβ1).…”

“…Human embryonic kidney 293T cells were transfected with an equimolar mixture of eight recombination substrates (TRBV1, 15, 16, 18, 20, 23, 24, and 26), pEBB-RAG1, and pEBB-RAG2, using Trans-IT 293 (Mirus) (40). Plasmid substrates were recovered 48 h posttransfection and digested with NotI to minimize unrearranged PCR products and DpnI to cut untransfected substrates (40).…”

“…Human embryonic kidney 293T cells were transfected with an equimolar mixture of eight recombination substrates (TRBV1, 15, 16, 18, 20, 23, 24, and 26), pEBB-RAG1, and pEBB-RAG2, using Trans-IT 293 (Mirus) (40). Plasmid substrates were recovered 48 h posttransfection and digested with NotI to minimize unrearranged PCR products and DpnI to cut untransfected substrates (40). The digested DNA mixture was amplified with primers that are common to all substrates-one that recognizes plasmid sequence upstream of the Vβs and one specific for Jβ1.1 (dsT7-CAAGCTGGCTAGCGTTTAAAC and J1.1TR-CTCGAATATGGACACGGAG GACATGC).…”

“…In brief, this algorithm calculates the theoretical recombination potential of an RSS using a statistical model that assigns a score based on the contribution of each nucleotide within the heptamer-spacer-nonamer sequence. The algorithm output is a recombination signal information content (RIC) score, which predicts the quality of an input RSS with a reasonable degree of accuracy based on data from plasmid recombination substrates (40). For Tcrb, 6 of the 35 Vβ gene segments are flanked by nonfunctional RSSs with a RIC score of <−58.5, the threshold defined by Cowell et al (39), (TRBV8, 12-3, 18, 21, 27, and 28).…”