Double-strand signal sequence breaks in V(D)J recombination are blunt, 5'-phosphorylated, RAG-dependent, and cell cycle regulated.

Schlissel, Mark S.; Constantinescu, Andreï; Morrow, T.; Baxter, MJ; Peng, Albert

doi:10.1101/gad.7.12b.2520

Cited by 360 publications

(359 citation statements)

References 51 publications

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“…This multistep process produces coding joints with considerable junction variability, which significantly contributes to the diversity of the immune repertoire. Signal ends are readily detectable and are persistent in cells undergoing V(D)J recombination, while coding ends are only detectable on limited occasions (7)(8)(9)(10). Thus, counterintuitively, the direct ligation of signal ends appears to be much slower than the multistep joining of coding ends.…”

mentioning

confidence: 90%

Metabolism of Recombination Coding Ends in scid Cells

Brown

Chang

2000

The Journal of Immunology

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V(D)J recombination cleavage generates two types of dsDNA breaks: blunt signal ends and covalently sealed hairpin coding ends. Although signal ends can be directly ligated to form signal joints, hairpin coding ends need to be opened and subsequently processed before being joined. However, the underlying mechanism of coding end resolution remains undefined. The current study attempts to delineate this process by analyzing various structures of coding ends made in situ from recombination-inducible pre-B cell lines of both normal and scid mice. These cell lines were derived by transformation of B cell precursors with the temperature-sensitive Abelson murine leukemia virus. Our kinetic analysis revealed that under conditions permissive to scid transformants, hairpin coding ends could be nicked to generate 3′ overhangs and then processed into blunt ends. The final joining of these blunt ends followed the same kinetics as signal joint formation. The course of this process is in sharp contrast to coding end resolution in scid heterozygous transformants that express the catalytic subunit of DNA-dependent protein kinase, in which hairpin end opening, processing, and joining proceeded very rapidly and appeared to be closely linked. Furthermore, we demonstrated that the opening of hairpin ends in scid cells could be manipulated by different culture conditions, which ultimately influenced not only the level and integrity of the newly formed coding joints, but also the extent of microhomology at the coding junctions. These results are discussed in the context of scid leaky recombination.

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mentioning

confidence: 90%

Metabolism of Recombination Coding Ends in scid Cells

Brown

Chang

2000

The Journal of Immunology

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“…RAG-2 was also expressed in the largely non-edited B cells derived from CD22 -/-D42H/Vj8-Jj5-Tg mice. In order to look at ongoing rearrangements more directly, we used a ligation-mediated PCR (LM-PCR) to detect signal break ends resulting from RAG- mediated DNA cleavage [34]. In this experiment, the level of secondary DNA rearrangements in purified splenic B cells from H/L double-Tg mice was compared with that of 493 + BM B cells (excluding recirculating mature B cells and plasma cells) from the same mice (Fig.…”

Section: Peripheral Receptor Editing In H/l Doubletransgenic Cd22-defmentioning

confidence: 99%

“…LM-PCR was carried out essentially as described by Schlissel et al [34]. Following ligation, a semi-quantitative radioactive PCR was carried out as described above with a Jj1-specific forward primer 5 0 -TTCCACGCATGCTTGGAGAG-3 0 and the reverse primer 5 0 -CCGGGAGATCTGAATTCAC-3 0 [34], resulting in a 130-bp product.…”

Section: Ligation-mediated Pcr and Rag-2 Expressionmentioning

confidence: 99%

“…Following ligation, a semi-quantitative radioactive PCR was carried out as described above with a Jj1-specific forward primer 5 0 -TTCCACGCATGCTTGGAGAG-3 0 and the reverse primer 5 0 -CCGGGAGATCTGAATTCAC-3 0 [34], resulting in a 130-bp product. Cj PCR amplification (90-bp DNA product) was used for quantification of B cell DNA.…”

Section: Ligation-mediated Pcr and Rag-2 Expressionmentioning

confidence: 99%

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Peripheral B cell receptor editing may promote the production of high‐affinity autoantibodies in CD22‐deficient mice

et al. 2006

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CD22-deficient mice are characterized by B cell hyperactivity and autoimmunity. We have constructed knock-in CD22 -/-mice, expressing an anti-DNA heavy (H) chain (D42), alone or combined with Vj1-Jj1 or Vj8-Jj5 light (L) chains. The Ig-targeted mice produced a lupus-like serology that was age-and sex-dependent. High-affinity IgG autoantibodies were largely dependent on the selection of B cells with a particular H/L combination, in which a non-transgenic, endogenous L chain was assembled by secondary rearrangements through the mechanism of receptor editing. Moreover, we present evidence that these secondary rearrangements are very prominent in splenic peripheral B cells. Since CD22 is primarily expressed on the surface of peripheral B cells, we propose a model for the development of a lupus-like autoimmune disease by a combination of peripheral receptor editing and abnormal B cell activation. IntroductionSystemic lupus erythematosus (SLE) is a rheumatic disease of unknown etiology, usually occurring in young women of childbearing age. The presence of serum antibodies to a variety of self components and their possible involvement in the development of severe kidney disease (glomerulonephritis) has made SLE a prototype of systemic autoimmune diseases [1]. The autoantibodies in lupus sera react with a large variety of nuclear antigens, including DNA, RNA and nuclear proteins. The antibodies to dsDNA are mostly highaffinity IgG that appear almost exclusively in SLE and very rarely in other diseases [2, 3].Several mouse models which spontaneously develop a lupus-like disease have been studied extensively [4]. The NZB/NZW F1 mouse is considered to be the murine model most closely resembling human SLE [5]. The lupus-like disease in these mice is more severe in females and is accompanied by high-affinity IgG anti-dsDNA autoantibodies. Both NZB and NZW parents contribute multiple susceptibility genes to the immune abnormalities of the F1 hybrid mouse [6]. Additional mouse models of SLE include mouse strains with deficiencies in antigen disposal mechanisms and mice that are deficient in proteins regulating the thresholds for tolerance and activation of B and T lymphocytes [7].CD22 is a B cell-specific surface glycoprotein of the Ig superfamily expressed on mature B cells [8, 9]. It functions as an adhesion molecule, as a signal-transducing molecule and as a modulator of intracellular signaling through the B cell receptor (BCR) complex. Negative regulatory roles for CD22 in BCR signaling are proposed to be critical for normal B cell activation, since immunoreceptor tyrosine-based inhibitory motifs with- in CD22 recruit SHP-1, a potent intracellular phosphatase with inhibitory functions in BCR signaling [10,11]. CD22-deficient mice [12][13][14][15] were reported to have decreased numbers of circulating B cells and slightly increased numbers of peritoneal B-1 cells, as well as higher levels of serum IgM [12,14]. Significantly, mature B cells from CD22-deficient mice had decreased expression of cell surface IgM and augmented i...

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“…Ligation-mediated PCR has been described in detail previously (17,30). Briefly, to measure T cell-specific intermediates in TCR␤ V to DJ recombination, linkers were ligated to genomic DNA isolated as described above.…”

Section: Detection Of Rag Gene Expression and V(d)j Recombination Intmentioning

confidence: 99%

T Cell Receptor Revision Does Not Solely Target Recent Thymic Emigrants

Cooper

Orr

McMahan

et al. 2003

The Journal of Immunology

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CD4+Vβ5+ T cells enter one of two tolerance pathways after recognizing a peripherally expressed superantigen encoded by an endogenous retrovirus. One pathway leads to deletion, while the other, termed TCR revision, results in cellular rescue upon expression of an alternate TCR that no longer recognizes the tolerogen. TCR revision requires the rearrangement of novel TCR β-chain genes and depends on recombinase-activating gene (RAG) expression in peripheral T cells. In line with recent findings that RAG+ splenic B cells are immature cells that have maintained RAG expression, it has been hypothesized that TCR revision is limited to recent thymic emigrants that have maintained RAG expression and TCR loci in a recombination-permissive configuration. Using mice in which the expression of green fluorescent protein is driven by the RAG2 promoter, we now show that in vitro stimulation can drive reporter expression in noncycling, mature, peripheral CD4+ T cells. In addition, thymectomized Vβ5 transgenic RAG reporter mice are used to demonstrate that TCR revision can target peripheral T cells up to 2 mo after thymectomy. Both sets of experiments strongly suggest that reinduction of RAG genes triggers TCR revision. Approximately 3% of CD4+Vβ5+ T cells in thymectomized Vβ5 transgenic reporter mice have undergone TCR revision within the previous 4–5 days. TCR revision can also occur in Vβ5+ T cells from nontransgenic mice, illustrating the relevance of this novel tolerance mechanism in unmanipulated animals.

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Double-strand signal sequence breaks in V(D)J recombination are blunt, 5'-phosphorylated, RAG-dependent, and cell cycle regulated.

Cited by 360 publications

References 51 publications

Metabolism of Recombination Coding Ends in scid Cells

Metabolism of Recombination Coding Ends in scid Cells

Peripheral B cell receptor editing may promote the production of high‐affinity autoantibodies in CD22‐deficient mice

T Cell Receptor Revision Does Not Solely Target Recent Thymic Emigrants

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