scholarly journals Control of V(D)J Recombination through Transcriptional Elongation and Changes in Locus Chromatin Structure and Nuclear Organization

2011 ◽  
Vol 2011 ◽  
pp. 1-10 ◽  
Author(s):  
Beatriz del Blanco ◽  
Vanina García ◽  
Alberto García-Mariscal ◽  
Cristina Hernández-Munain
Keyword(s):  
Chromatin Structure ◽  
Antigen Receptor ◽  
Open Chromatin ◽  
Recombination Reaction ◽  
Transcriptional Elongation ◽  
Signal Sequences ◽  
Recombination Signal ◽  
Recombination Signal Sequences ◽  
Rag 1 ◽  
Antigen Receptor Loci

V(D)J recombination is the assembly of gene segments at the antigen receptor loci to generate antigen receptor diversity in T and B lymphocytes. This process is regulated, according to defined developmental programs, by the action of a single specific recombinase complex formed by the recombination antigen gene (RAG-1/2) proteins that are expressed in immature lymphocytes. V(D)J recombination is strictly controlled by RAG-1/2 accessibility to specific recombination signal sequences in chromatin at several levels: cellular lineage, temporal regulation, gene segment order, and allelic exclusion. DNA cleavage by RAG-1/2 is regulated by the chromatin structure, transcriptional elongation, and three-dimensional architecture and position of the antigen receptor loci in the nucleus. Cis-elements specifically direct transcription and V(D)J recombination at these loci through interactions with transacting factors that form molecular machines that mediate a sequence of structural events. These events open chromatin to activate transcriptional elongation and to permit the access of RAG-1/2 to their recombination signal sequences to drive the juxtaposition of the V, D, and J segments and the recombination reaction itself. This chapter summarizes the advances in this area and the important role of the structure and position of antigen receptor loci within the nucleus to control this process.

scholarly journals Footprint Analysis of Recombination Signal Sequences in the 12/23 Synaptic Complex of V(D)J Recombination

2002 ◽  
Vol 22 (20) ◽  
pp. 7217-7225 ◽  
Author(s):  
Fumikiyo Nagawa ◽  
Masami Kodama ◽  
Tadashi Nishihara ◽  
Kei-ichiro Ishiguro ◽  
Hitoshi Sakano
Keyword(s):  
Magnetic Beads ◽  
Antigen Receptor ◽  
Dnase I ◽  
Signal Sequences ◽  
Synaptic Complex ◽  
Recombination Signal ◽  
Recombination Activating Genes ◽  
Recombination Signal Sequences ◽  
Dnase I Footprinting ◽  
Footprint Analysis

ABSTRACT In V(D)J joining of antigen receptor genes, two recombination signal sequences (RSSs), 12-RSS and 23-RSS, are paired and complexed with the protein products of recombination-activating genes RAG1 and RAG2. Using magnetic beads, we purified the pre- and postcleavage complexes of V(D)J joining and analyzed them by DNase I footprinting. In the precleavage synaptic complex, strong protection was seen not only in the 9-mer and spacer regions but also near the coding border of the 7-mer. This is a sharp contrast to the single RSS-RAG complex where the 9-mer plays a major role in the interaction. We also analyzed the postcleavage signal end complex by footprinting. Unlike what was seen with the precleavage complex, the entire 7-mer and its neighboring spacer regions were protected. The present study indicates that the RAG-RSS interaction in the 7-mer region drastically changes once the synaptic complex is formed for cleavage.

scholarly journals High Throughput Characterization of V(D)J Recombination Signal Sequences Redefines the Consensus Sequence

2021 ◽  
Author(s):  
Walker Hoolehan ◽  
Justin C. Harris ◽  
Jennifer N. Byrum ◽  
Karla K. Rodgers
Keyword(s):  
Active Site ◽  
Receptor Gene ◽  
Consensus Sequence ◽  
Antigen Receptor ◽  
Massively Parallel ◽  
Signal Sequences ◽  
Recombination Signal ◽  
Recombination Signal Sequences ◽  
Purine Pyrimidine

ABSTRACTIn the adaptive immune system, V(D)J recombination initiates the production of a diverse antigen receptor repertoire in developing B and T cells. Recombination activating proteins, RAG1 and RAG2 (RAG1/2), catalyze V(D)J recombination by cleaving adjacent to recombination signal sequences (RSSs) that flank antigen receptor gene segments. Previous studies defined the consensus RSS as containing conserved heptamer and nonamer sequences separated by a less conserved 12 or 23 base-pair spacer sequence. However, many RSSs deviate from the consensus sequence. Here, we developed a cell-based, massively parallel V(D)J recombination assay to evaluate RAG1/2 activity on thousands of RSSs. We focused our study on the RSS heptamer and adjoining spacer region, as this region undergoes extensive conformational changes during RAG-mediated DNA cleavage. While the consensus heptamer sequence (CACAGTG) was marginally preferred, RAG1/2 was highly active on a wide range of non-consensus sequences. RAG1/2 generally preferred select purine/pyrimidine motifs that may accommodate heptamer unwinding in the RAG1/2 active site. Our results suggest RAG1/2 specificity for RSS heptamers is primarily dictated by DNA structural features dependent on purine/pyrimidine pattern, and to a lesser extent, RAG:RSS base-specific interactions. Further investigation of RAG1/2 specificity using this new approach will help elucidate the genetic instructions guiding V(D)J recombination.Summary StatementPartially conserved recombination signal sequences (RSSs) govern antigen receptor gene assembly during V(D)J recombination. Here, a massively parallel analysis of randomized RSSs reveals key attributes that allow DNA sequence diversity in the RAG1/2 active site and that contribute to the differential utilization of RSSs in endogenous V(D)J recombination. Overall, these results will assist identification of RAG1/2 off-target sites, which can drive leukemia cell transformation, as well as characterization of bona fide RSSs used to generate antigen receptor diversity.

scholarly journals RSSsite: a reference database and prediction tool for the identification of cryptic Recombination Signal Sequences in human and murine genomes

2010 ◽  
Vol 38 (Web Server) ◽  
pp. W262-W267 ◽  
Author(s):  
I. Merelli ◽  
A. Guffanti ◽  
M. Fabbri ◽  
A. Cocito ◽  
L. Furia ◽  
...  
Keyword(s):  
Reference Database ◽  
Prediction Tool ◽  
Signal Sequences ◽  
Recombination Signal ◽  
Recombination Signal Sequences

scholarly journals Restrictions Limiting the Generation of DNA Double Strand Breaks during Chromosomal V(D)J Recombination

2002 ◽  
Vol 195 (3) ◽  
pp. 309-316 ◽  
Author(s):  
Robert E. Tillman ◽  
Andrea L. Wooley ◽  
Maureen M. Hughes ◽  
Tara D. Wehrly ◽  
Wojciech Swat ◽  
...  
Keyword(s):  
Dna Cleavage ◽  
Receptor Gene ◽  
Antigen Receptor ◽  
Double Strand Breaks ◽  
Recombination Reaction ◽  
Dna Double Strand Breaks ◽  
Strand Breaks ◽  
Recombination Signal Sequences ◽  
Dna Dsbs ◽  
Cleavage Step

Antigen receptor loci are composed of numerous variable (V), diversity (D), and joining (J) gene segments, each flanked by recombination signal sequences (RSSs). The V(D)J recombination reaction proceeds through RSS recognition and DNA cleavage steps making it possible for multiple DNA double strand breaks (DSBs) to be introduced at a single locus. Here we use ligation-mediated PCR to analyze DNA cleavage intermediates in thymocytes from mice with targeted RSS mutations at the endogenous TCRβ locus. We show that DNA cleavage does not occur at individual RSSs but rather must be coordinated between RSS pairs flanking gene segments that ultimately form coding joins. Coordination of the DNA cleavage step occurs over great distances in the chromosome and favors intra- over interchromosomal recombination. Furthermore, through several restrictions imposed on the generation of both nonpaired and paired DNA DSBs, this requirement promotes antigen receptor gene integrity and genomic stability in developing lymphocytes undergoing V(D)J recombination.

scholarly journals The role of components of recombination signal sequences in immunoglobulin gene segment usage: a V81x model

1999 ◽  
Vol 27 (11) ◽  
pp. 2304-2309 ◽  
Author(s):  
M. Larijani ◽  
C. C. K. Yu ◽  
R. Golub ◽  
Q. L. K. Lam ◽  
G. E. Wu
Keyword(s):  
Gene Segment ◽  
Immunoglobulin Gene ◽  
Signal Sequences ◽  
Recombination Signal ◽  
Recombination Signal Sequences
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