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 Footprint Analysis of the RAG Protein Recombination Signal Sequence Complex for V(D)J Type Recombination

1998 ◽  
Vol 18 (1) ◽  
pp. 655-663 ◽  
Author(s):  
Fumikiyo Nagawa ◽  
Kei-ichiro Ishiguro ◽  
Akio Tsuboi ◽  
Tomoyuki Yoshida ◽  
Akiko Ishikawa ◽  
...  
Keyword(s):  
Signal Sequence ◽  
Dimethyl Sulfate ◽  
Dnase I ◽  
Mobility Shift ◽  
Partial Methylation ◽  
Recombination Signal ◽  
Recombination Activating Genes ◽  
Bottom Strand ◽  
Dnase I Footprinting ◽  
Gel Mobility Shift

ABSTRACT We have studied the interaction between recombination signal sequences (RSSs) and protein products of the truncated forms of recombination-activating genes (RAG) by gel mobility shift, DNase I footprinting, and methylation interference assays. Methylation interference with dimethyl sulfate demonstrated that binding was blocked by methylation in the nonamer at the second-position G residue in the bottom strand and at the sixth- and seventh-position A residues in the top strand. DNase I footprinting experiments demonstrated that RAG1 alone, or even a RAG1 homeodomain peptide, gave footprint patterns very similar to those obtained with the RAG1-RAG2 complex. In the heptamer, partial methylation interference was observed at the sixth-position A residue in the bottom strand. In DNase I footprinting, the heptamer region was weakly protected in the bottom strand by RAG1. The effects of RSS mutations on RAG binding were evaluated by DNA footprinting. Comparison of the RAG-RSS footprint data with the published Hin model confirmed the notion that sequence-specific RSS-RAG interaction takes place primarily between the Hin domain of the RAG1 protein and adjacent major and minor grooves of the nonamer DNA.

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 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 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

scholarly journals Synapsis of Recombination Signal Sequences Located in cis and DNA Underwinding in V(D)J Recombination

2004 ◽  
Vol 24 (19) ◽  
pp. 8727-8744 ◽  
Author(s):  
Mihai Ciubotaru ◽  
David G. Schatz
Keyword(s):  
Conformational Changes ◽  
Dna Cleavage ◽  
High Mobility ◽  
Experimental System ◽  
High Mobility Group Protein ◽  
Signal Sequences ◽  
Recombination Signal ◽  
Recombination Signal Sequences ◽  
Group Protein ◽  
Dna Substrates

ABSTRACT V(D)J recombination requires binding and synapsis of a complementary (12/23) pair of recombination signal sequences (RSSs) by the RAG1 and RAG2 proteins, aided by a high-mobility group protein, HMG1 or HMG2. Double-strand DNA cleavage within this synaptic, or paired, complex is thought to involve DNA distortion or melting near the site of cleavage. Although V(D)J recombination normally occurs between RSSs located on the same DNA molecule (in cis), all previous studies that directly assessed RSS synapsis were performed with the two DNA substrates in trans. To overcome this limitation, we have developed a facilitated circularization assay using DNA substrates of reduced length to assess synapsis of RSSs in cis. We show that a 12/23 pair of RSSs is the preferred substrate for synapsis of cis RSSs and that the efficiency of pairing is dependent upon RAG1-RAG2 stoichiometry. Synapsis in cis occurs rapidly and is kinetically favored over synapsis of RSSs located in trans. This experimental system also allowed the generation of underwound DNA substrates containing pairs of RSSs in cis. Importantly, we found that the RAG proteins cleave such substrates substantially more efficiently than relaxed substrates and that underwinding may enhance RSS synapsis as well as RAG1/2-mediated catalysis. The energy stored in such underwound substrates may be used in the generation of DNA distortion and/or protein conformational changes needed for synapsis and cleavage. We propose that this unwinding is uniquely sensed during synapsis of an appropriate 12/23 pair of RSSs.

Recombination signal sequences restrict chromosomal V(D)J recombination beyond the 12/23 rule

Nature ◽  
2000 ◽  
Vol 405 (6786) ◽  
pp. 583-586 ◽  
Author(s):  
Craig H. Bassing ◽  
Frederick W. Alt ◽  
Maureen M. Hughes ◽  
Margaux D'Auteuil ◽  
Tara D. Wehrly ◽  
...  
Keyword(s):  
Signal Sequences ◽  
Recombination Signal ◽  
Recombination Signal Sequences
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