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PubMed2001

Effect of HIV integrase inhibitors on the RAG1/2 recombinase.

Assembly of functional Ig and T cell receptor genes by V(D)J recombination depends on site-specific cleavage of chromosomal DNA by the RAG1/2 recombinase. As RAG1/2 action has mechanistic similarities to DNA transposases and integrases such as HIV-1 integrase, we sought to determine how integrase inhibitors of the diketo acid type would affect the various activities of RAG1/2. Both of the inhibitors we tested interfered with DNA cleavage and disintegration activities of RAG1/2, apparently by disrupting interaction with the DNA motifs bound specifically by the recombinase. The inhibitors did not ablate RAG1/2's transposition activity or capture of nonspecific transpositional target DNA, suggesting this DNA occupies a site on the recombinase different from that used for specific binding. These results further underscore the similarities between RAG1/2 and integrase and suggest that certain integrase inhibitors may have the potential to interfere with aspects of B and T cell development.

Amino Acid Motifs↗

PubMed2003

In vivo transposition mediated by V(D)J recombinase in human T lymphocytes.

The rearrangement of immunoglobulin (Ig) and T-cell receptor (TCR) genes in lymphocytes by V(D)J recombinase is essential for immunological diversity in humans. These DNA rearrangements involve cleavage by the RAG1 and RAG2 (RAG1/2) recombinase enzymes at recombination signal sequences (RSS). This reaction generates two products, cleaved signal ends and coding ends. Coding ends are ligated by non-homologous end-joining proteins to form a functional Ig or TCR gene product, while the signal ends form a signal joint. In vitro studies have demonstrated that RAG1/2 are capable of mediating the transposition of cleaved signal ends into non-specific sites of a target DNA molecule. However, to date, in vivo transposition of signal ends has not been demonstrated. We present evidence of in vivo inter-chromosomal transposition in humans mediated by V(D)J recombinase. T-cell isolates were shown to contain TCRalpha signal ends from chromosome 14 inserted into the X-linked hypo xanthine-guanine phosphoribosyl transferase locus, resulting in gene inactivation. These findings implicate V(D)J recombinase-mediated transposition as a mutagenic mechanism capable of deleterious genetic rearrangements in humans.

Base Sequence↗

PubMed2001

Intermediates in V(D)J recombination: a stable RAG1/2 complex sequesters cleaved RSS ends.

Rearrangement of gene segments to generate antigen receptor coding regions depends on the RAG1/2 recombinase, which assembles a synaptic complex between two DNA signal sequences and then cleaves the DNA directly adjacent to the paired signals. After coupled cleavage of complementary signal sequences, virtually all of the cleaved signal ends remained associated with RAG1/2 in stable complexes. These signal end complexes were distinct from various precleavage RAG1/2 signal complexes in that they were resistant to treatment with heparin. A mammalian joining apparatus consisting of purified Ku70/86, XRCC4, and DNA ligase IV proteins was sufficient to join deproteinized cleaved ends, but retention of signal sequences within the signal end complex blocked access to the DNA ends and prevented their joining by these proteins. Sequestration of cleaved ends within the signal end complex would account for the persistence of these ends in the cell after cleavage and may explain why they do not normally activate the DNA-damage-dependent cell cycle checkpoint.

Antigens, Nuclear↗

PubMed2004

Involvement of V(D)J recombinase in the generation of intragenic deletions in the Rit1/Bcl11b tumor suppressor gene in gamma-ray-induced thymic lymphomas and in normal thymus of the mouse.

Mouse thymic lymphomas induced by gamma-irradiation exhibited homozygous deletions of the Rit1/Bcl11b tumor suppressor gene on chromosome 12 at high frequencies. Internal deletions of one allele were frequently accompanied by loss of the other allele. In order to elucidate the mechanism of these internal deletions, the sites of breakage and rejoining were examined by PCR mapping and sequencing. The 5' site of the deletions clustered within an approximately 5 kb region of intron 1 and the 3' site was confined to a site in intron 3. These sites contained P and/or N nucleotides and cryptic sequences recognizable by the RAG1/2 recombinase in the vicinity. This suggests that the Rit1 intragenic deletions were generated by endogenous illegitimate V(D)J recombinase activity and such aberrant recombination was also detected by nested PCR of DNA from the thymus of unirradiated mice but not of RAG2-deficient mice. A rough estimate indicated that there reside as many as 10(3)-10(4) thymocytes having Rit1 deletions, assuming the presence of 10(8) thymocytes in the thymus of unirradiated mice. Moreover, the recombination frequency was not affected by gamma-irradiation. These results show no effect of radiation on Rit1 mutations and suggest an indirect mechanism for its role in lymphomagenesis.

Animals↗

PubMed2004

How to keep V(D)J recombination under control.

Breaking apart chromosomes is not a matter to be taken lightly. The possible negative outcomes are obvious: loss of information, unstable chromosomes, chromosomal translocations, tumorigenesis, or cell death. Utilizing DNA rearrangement to generate the desired diversity in the antigen receptor loci is a risky business, and it must be carefully controlled. In general, the regulation is so precise that the negative consequences are minimal or not apparent. They are visible only when the process of V(D)J recombination goes awry, as for example in some chromosomal translocations associated with lymphoid tumors. Regulation is imposed not only to prevent the generation of random breaks in the DNA, but also to direct rearrangement to the appropriate locus or subregion of a locus in the appropriate cell at the appropriate time. Antigen receptor rearrangement is regulated essentially at four different levels: expression of the RAG1/2 recombinase, intrinsic biochemical properties of the recombinase and the cleavage reaction, the post-cleavage /DNA repair stage of the process, and accessibility of the substrate to the recombinase. Within each of these broad categories, multiple mechanisms are used to achieve the desired aims. The major focus of this review is on accessibility control and the role of chromatin and nuclear architecture in achieving this regulation, although other issues are touched upon.

Alleles↗

PubMed2026

Characterization of a Ku-binding motif in the C-terminal region of RAG2.

We applied an unsupervised interactome analysis with the RAG2 C-terminal region (R2CT) in v-abl pro-B cells undergoing V(D)J recombination. Mass-spectrometry analyses showed that Ku70 and Ku80 were among the top 10 hits. To further strengthen these observations, we performed Proximity Ligation Assay (PLA) and characterize the existence of a GFP-R2CT-Ku complex formation in cellulo. The interaction of several partners with Ku70/80 (Ku) through Ku-binding motifs (KBMs) in their sequences governs their enrolment in NHEJ repair complexes. Through sequence analysis, we identified a KBM within R2CT (R-KBM, amino acids 589-527). We confirmed by calorimetry a specific micromolar interaction between this RAG2 region and Ku70/80/DNA complex. The RAG2 motif KBM can be subdivided in two conserved parts that have no interaction individually. AlphaFold2 prediction coupled with molecular dynamic simulations indicate that the C-terminal part of the RAG2 motif interacts with Ku80 on the same site than the NHEJ factor XLF. These in silico analyses indicated that the N-terminal part of the RAG2 motif interacts with DNA adjacent to Ku with a major role of the K503 residue in agreement with disruption of the interaction observed with the K503E mutant. This study further extends the large ensemble of proteins recruited at DSBs by KBM motifs and substantiates the model of a tight coupling between DNA breakage and repair during V(D)J recombination, mediated by the Ku-RAG2 C-terminus interaction.

Ku Autoantigen↗

PubMed2006

Target DNA structure plays a critical role in RAG transposition.

Antigen receptor gene rearrangements are initiated by the RAG1/2 protein complex, which recognizes specific DNA sequences termed RSS (recombination signal sequences). The RAG recombinase can also catalyze transposition: integration of a DNA segment bounded by RSS into an unrelated DNA target. For reasons that remain poorly understood, such events occur readily in vitro, but are rarely detected in vivo. Previous work showed that non-B DNA structures, particularly hairpins, stimulate transposition. Here we show that the sequence of the four nucleotides at a hairpin tip modulates transposition efficiency over a surprisingly wide (>100-fold) range. Some hairpin targets stimulate extraordinarily efficient transposition (up to 15%); one serves as a potent and specific transposition inhibitor, blocking capture of targets and destabilizing preformed target capture complexes. These findings suggest novel regulatory possibilities and may provide insight into the activities of other transposases.

Animals↗

PubMed2001

Identical mutations in RAG1 or RAG2 genes leading to defective V(D)J recombinase activity can cause either T-B-severe combined immune deficiency or Omenn syndrome.

Omenn syndrome (OS) is an inherited disorder characterized by an absence of circulating B cells and an infiltration of the skin and the intestine by activated oligoclonal T lymphocytes, indicating that a profound defect in the lymphoid developmental program could be accountable for this condition. Inherited mutations in either the recombination activating genes RAG1 or RAG2, resulting in partial V(D)J recombinase activity, were shown to be responsible for OS. This study reports on the characterization of new RAG1/2 gene mutations in a series of 9 patients with OS. Given the occurrence of the same mutations in patients with T-B-severe combined immune deficiency or OS on 3 separate occasions, the proposal is made that an additional factor may be required in certain circumstances for the development of the Omenn phenotype. The nature of this factor is discussed.

DNA Nucleotidyltransferases↗

PubMed2004

The taming of a transposon: V(D)J recombination and the immune system.

The genes that encode immunoglobulins and T-cell receptors must be assembled from the multiple variable (V), joining (J), and sometimes diversity (D) gene segments present in the germline loci. This process of V(D)J recombination is the major source of the immense diversity of the immune repertoire of jawed vertebrates. The recombinase that initiates the process, recombination-activating genes 1 (RAG1) and RAG2, belongs to a large family that includes transposases and retroviral integrases. RAG1/2 cleaves the DNA adjacent to the gene segments to be recombined, and the segments are then joined together by DNA repair factors. A decade of biochemical research on RAG1/2 has revealed many similarities to transposition, culminating with the observation that RAG1/2 can carry out transpositional strand transfer. Here, we discuss the parallels between V(D)J recombination and transposition, focusing specifically on the assembly of the recombination nucleoprotein complex, the mechanism of cleavage, the disassembly of post-cleavage complexes, and aberrant reactions carried out by the recombinase that do not result in successful locus rearrangement and may be deleterious to the organism. This work highlights the considerable diversity of transposition systems and their relation to V(D)J recombination.

DNA Repair↗

PubMed2006

SLP65 deficiency results in perpetual V(D)J recombinase activity in pre-B-lymphoblastic leukemia and B-cell lymphoma cells.

Perpetual V(D)J recombinase activity involving multiple DNA double-strand break events in B-cell lineage leukemia and lymphoma cells may introduce secondary genetic aberrations leading towards malignant progression. Here, we investigated defective negative feedback signaling through the (pre-) B-cell receptor as a possible reason for deregulated V(D)J recombinase activity in B-cell malignancy. On studying 28 cases of pre-B-lymphoblastic leukemia and 27 B-cell lymphomas, expression of the (pre-) B-cell receptor-related linker molecule SLP65 (SH2 domain-containing lymphocyte protein of 65 kDa) was found to be defective in seven and five cases, respectively. SLP65 deficiency correlates with RAG1/2 expression and unremitting V(H) gene rearrangement activity. Reconstitution of SLP65 expression in SLP65-deficient leukemia and lymphoma cells results in downregulation of RAG1/2 expression and prevents both de novo V(H)-DJ(H) rearrangements and secondary V(H) replacement. We conclude that iterative V(H) gene rearrangement represents a frequent feature in B-lymphoid malignancy, which can be attributed to SLP65 deficiency in many cases.

Adaptor Proteins, Signal Transducing↗

PubMed2006

Hypothesis: a biological role for germline transcription in the mechanism of V(D)J recombination--implications for initiation of allelic exclusion.

The sequences that encode the antigen-binding sites of IgH and IgL chains - variable (V), diversity (D, H chain loci only) and joining (J) sequences - are configured as separate DNA segments at the germline level. Expression of an Ig molecule requires V(D)J assembly. Productive V(D)J recombination is monoallelic. How rearrangement is initiated differentially at maternal and paternal alleles is unclear. The products of recombination activating gene (RAG)1 and RAG2 mediate rearrangement by cleaving the DNA between an unrearranged gene segment and adjacent recombination signal sequences (RSS). It is proposed that supercoiling generated during germline transcription at Ig loci (which occurs concomitantly with rearrangement) is required at RSS for RAG1/2 recognition. Rearrangement might hence initiate sequentially at maternal and paternal alleles where deregulated germline transcription causes RAG1/2 recognition of RSS to become stochastic.

Alleles↗

PubMed2002

Ordered assembly of the V(D)J synaptic complex ensures accurate recombination.

Recombination of gene segments at the immunoglobulin and T-cell receptor loci requires that the RAG1 and RAG2 proteins bring together DNA signal sequences (RSSs) with 12- and 23-bp spacers into a synaptic complex and cleave the DNA. A RAG1/2 multimer that can cleave both signals is shown to assemble on an isolated RSS, and the complementary RSS enters this complex as naked DNA. When RAG1/2 is allowed to bind 12 and 23 RSSs separately prior to their mixing, synaptic complex assembly and cleavage activity are greatly reduced, indicating that only a complex initially assembled on a single RSS leads to productive cleavage. RAG1/2 complexes assembled on 12 RSSs will only incorporate 23 partners, while complexes assembled on 23 RSSs show a 5- to 6-fold preference for 12 partners. Thus, initial assembly on a 12 RSS most accurately reflects the strict 12/23 coupled cleavage observed in the cell. Additional cellular factors such as chromatin may ensure that RAG1/2 first assembles on a 12 RSS, and then a free 23 RSS enters to activate cleavage.

Animals↗

PubMed2005

Determinants of HMGB proteins required to promote RAG1/2-recombination signal sequence complex assembly and catalysis during V(D)J recombination.

Efficient assembly of RAG1/2-recombination signal sequence (RSS) DNA complexes that are competent for V(D)J cleavage requires the presence of the nonspecific DNA binding and bending protein HMGB1 or HMGB2. We find that either of the two minimal DNA binding domains of HMGB1 is effective in assembling RAG1/2-RSS complexes on naked DNA and stimulating V(D)J cleavage but that both domains are required for efficient activity when the RSS is incorporated into a nucleosome. The single-domain HMGB protein from Saccharomyces cerevisiae, Nhp6A, efficiently assembles RAG1/2 complexes on naked DNA; however, these complexes are minimally competent for V(D)J cleavage. Nhp6A forms much more stable DNA complexes than HMGB1, and a variety of mutations that destabilize Nhp6A binding to bent microcircular DNA promote increased V(D)J cleavage. One of the two DNA bending wedges on Nhp6A and the analogous phenylalanine wedge at the DNA exit site of HMGB1 domain A were found to be essential for promoting RAG1/2-RSS complex formation. Because the phenylalanine wedge is required for specific recognition of DNA kinks, we propose that HMGB proteins facilitate RAG1/2-RSS interactions by recognizing a distorted DNA structure induced by RAG1/2 binding. The resulting complex must be sufficiently dynamic to enable the series of RAG1/2-mediated chemical reactions on the DNA.

Amino Acid Sequence↗

PubMed2000

The scid recombination-inducible cell line: a model to study DNA-PK-independent V(D)J recombination.

To investigate the molecular mechanisms of the variable (diversity) joining (V(D)J) recombination process at an endogenous gene locus, recombination-inducible cell lines were made from both bcl-2-bearing severe combined immune deficiency (scid) homozygous and scid heterozyous (s/ + ) mice by transforming pre-B cells with the temperature-sensitive Abelson murine leukemia virus (ts-Ab-MLV). These transformants can be induced to undergo immunoglobulin light-chain gene rearrangements by incubating them at the non-permissive temperature. In the case of transformed scid cells, a significant amount of hairpin coding ends are accumulated during recombination induction, but few coding joints are generated. After being shifted to the permissive temperature. however, these cells are capable of opening hairpin ends and forming coding joints. Thus, ts-Ab-MLV transformed scid cells can be readily manipulated for both recombination cleavage and end resolution. However, unlike the rapid coding joint formation in s/ + cells that have the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs), the process for resolving coding ends in scid cells is slow and error prone, and also appears to be correlated with a reduction in the RAG1/2 expression. Apparently, this process is mediated by a DNA-PK-independent pathway. The fact that the activity of this pathway can be manipulated in vitro makes it possible to delineate the mechanisms in end opening, processing and joining. Therefore, these ts-Ab-MLV transformed scid cell lines offer a model to study the molecular nature as well as the regulation of the DNA-PK-independent pathway in coding end resolution.

Animals↗

PubMed2002

RAG1-DNA binding in V(D)J recombination. Specificity and DNA-induced conformational changes revealed by fluorescence and CD spectroscopy.

The RAG1 and RAG2 proteins together constitute the nuclease that initiates the assembly of immunoglobulin and T cell receptor genes in a reaction known as V(D)J recombination. RAG1 plays a central role in recognition of the recombination signal sequence (RSS) by the RAG1/2 complex. To investigate the parameters governing the RAG1-RSS interaction, the murine core RAG1 protein (amino acids 377-1008) fused to a short Strep tag has been purified to homogeneity from bacteria. The Strep-RAG1 (StrRAG1) protein exists as a dimer at a wide range of protein concentrations (25-500 nM) in the absence of DNA and binds with reasonably high affinity and specificity (apparent K(D) = 41 nM) to the RSS. Both electrophoretic mobility shift assays and polarization anisotropy experiments indicate that only a single StrRAG1-DNA species exists in solution. Anisotropy decay measured by frequency domain spectroscopy suggests that the complex contains a dimer of StrRAG1 bound to a single DNA molecule. Using measurements of protein intrinsic fluorescence and circular dichroism, we demonstrate that StrRAG1 undergoes a major conformational change upon binding the RSS. Steady-state fluorescence and acrylamide quenching studies reveal that this conformational change is associated with a repositioning of intrinsic protein fluorophores from a hydrophobic to a solvent-exposed environment. RSS-induced conformational changes of StrRAG1 may influence the interaction of RAG1 with RAG2 and synaptic complex formation.

Animals↗