Intermolecular V(D)J Recombination Is Prohibited Specifically at the Joining Step

Han, Jung-Ok; Steen, Sharri Bockheim; Roth, David B.

doi:10.1016/s1097-2765(00)80460-8

Cited by 22 publications

(22 citation statements)

References 34 publications

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“…Finally, it was reported that 12-and 23-RSSs on separate transiently transfected plasmids could mediate relatively efficient synapsis, coupled cleavage, and signal joint formation. This study also reported a specific and dramatic defect in coding joint formation if the two RSSs were in trans, which led to the proposal that mechanisms exist that selectively disfavor or inhibit coding joint formation in trans (26).…”

mentioning

confidence: 73%

“…1) could be used to amplify joints for both the cis and trans configurations. This strategy is essentially identical to that employed in a recent analysis of the same issue (26). We also explored the effect of substrate topology by transfecting supercoiled, relaxed circular, and linearized forms of the substrates.…”

Section: Cis Versus Trans and The Effect Of Substrate Topological Stamentioning

confidence: 99%

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Intermolecular V(D)J Recombination

Tevelev¹,

Schatz

2000

Journal of Biological Chemistry

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V(D)J recombination plays a prominent role in the generation of the antigen receptor repertoires of B and T lymphocytes. It is also likely to be involved in the formation of chromosomal translocations, some of which may result from interchromosomal recombination. We have investigated the potential of the V(D)J recombination machinery to perform intermolecular recombination between two plasmids, either unlinked or linked by catenation. In either case, recombination occurs in trans to yield signal and coding joints, and the results do not support the existence of a mechanistic block to the formation of coding joints in trans. Instead, we observe that linearization of the substrate, which does not alter the cis or trans status of the recombination signals, causes a specific and dramatic reduction in coding joint formation. This unexpected result leads us to propose a "release and recapture" model for V(D)J recombination in which coding ends are frequently released from the postcleavage complex and the efficiency of coding joint formation is influenced by the efficiency with which such ends are recaptured by the complex. This implies the existence of mechanisms, operative during recombination of chromosomal substrates, that act to prevent coding end release or to facilitate coding end recapture.V(D)J recombination is the process of assembly of T-cell receptor and immunoglobulin genes from V, J, and sometimes D gene segments (1). Lymphoid-specific proteins RAG1 (recombination activating gene 1) and RAG2 (2, 3) and multiple ubiquitously expressed protein factors are essential for V(D)J recombination. RAG1 and RAG2 bind to and cleave at specific recombination signal sequences (RSSs) 1 generating blunt signal ends and covalently sealed, hairpin coding ends (4 -7). RSSs consist of conserved heptamer and nonamer sequences separated by a nonconserved spacer 12 or 23 base pairs long (12-and 23-RSS). V(D)J recombination occurs efficiently in vivo only between RSSs with different spacer lengths, a restriction known as the 12/23 rule (8).Recent biochemical studies indicate that V(D)J recombination proceeds through a series of protein-DNA complexes. Prior to cleavage, the RAG1 and RAG2 proteins form stable complexes with individual and synapsed pairs of RSSs (9 -17). After cleavage, the RAG proteins remain tightly bound to a synapsed pair of signal ends (15, 18) and also appear to interact with the two hairpin coding ends, albeit with lower affinity (15). Therefore, the immediate product of cleavage is thought to be a "cleaved signal complex" containing the four free ends (15).Usually, RSSs involved in V(D)J recombination are located on the same chromosome, i.e. in cis. However, V(D)J recombination has been implicated in the formation of chromosomal translocations leading to lymphoid malignancies (19 -23). In certain cases, such translocations could be the result of V(D)J recombination occurring in trans, using RSSs on different chromosomes. There have been attempts to address this issue. The first such study attempted, and fai...

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confidence: 73%

Section: Cis Versus Trans and The Effect Of Substrate Topological Stamentioning

confidence: 99%

Intermolecular V(D)J Recombination

Tevelev¹,

Schatz

2000

Journal of Biological Chemistry

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“…prohibited at the joining step (26). On the other hand, by increasing the local concentrations of two separate recombination substrates, Tevelev and Schatz have observed a higher level of intermolecular joining products (50).…”

Section: Vol 20 2000 Formation Of Ij and Hj Induced By V(d)j Recombmentioning

confidence: 99%

Activation of V(D)J Recombination Induces the Formation of Interlocus Joints and Hybrid Joints in scid Pre-B-Cell Lines

Lew

Franco

Chang

2000

Molecular and Cellular Biology

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V(D)J recombination is the mechanism by which antigen receptor genes are assembled. The site-specific cleavage mediated by RAG1 and RAG2 proteins generates two types of double-strand DNA breaks: blunt signal ends and covalently sealed hairpin coding ends. Although these DNA breaks are mainly resolved into coding joints and signal joints, they can participate in a nonstandard joining process, forming hybrid and open/shut joints that link coding ends to signal ends. In addition, the broken DNA molecules excised from different receptor gene loci could potentially be joined to generate interlocus joints. The interlocus recombination process may contribute to the translocation between antigen receptor genes and oncogenes, leading to malignant transformation of lymphocytes. To investigate the underlying mechanisms of these nonstandard recombination events, we took advantage of recombination-inducible cell lines derived from scid homozygous (s/s) and scid heterozygous (s/؉) mice by transforming B-cell precursors with a temperature-sensitive Abelson murine leukemia virus mutant (ts-Ab-MLV). We can manipulate the level of recombination cleavage and end resolution by altering the cell culture temperature. By analyzing various recombination products in scid and s/؉ ts-Ab-MLV transformants, we report in this study that scid cells make higher levels of interlocus and hybrid joints than their normal counterparts. These joints arise concurrently with the formation of intralocus joints, as well as with the appearance of opened coding ends. The junctions of these joining products exhibit excessive nucleotide deletions, a characteristic of scid coding joints. These data suggest that an inability of scid cells to promptly resolve their recombination ends exposes the ends to a random joining process, which can conceivably lead to chromosomal translocations.Developing lymphocytes have the unique ability to generate diverse antigen receptor molecules, immunoglobulins, and T-cell receptors. The assembly of these receptor genes is achieved through site-specific recombination events from separately encoded gene segments, variable (V), diversity (D), and joining (J) regions, in a process known as V(D)J recombination (6). Each gene segment is flanked by conserved recombination signal sequences (RSS) with a spacer of 12 or 23 nucleotides (12-RSS or 23-RSS, respectively). V(D)J recombination, catalyzed by enzymes encoded by recombination-activating genes 1 and 2 (RAG1 and RAG2), takes place at the junctions between RSS and coding gene segments (5, 19). Cleavage occurs coordinately at 12-RSS and 23-RSS, in accordance with what is known as the 12/23 rule. This site-specific cleavage generates two types of broken DNA molecules: bluntopened signal ends and covalently sealed coding ends (22, 39).The joining of these ends to form signal joints (SJ) and coding joints (CJ) is mediated by nonhomologous end joining machinery, which is believed to be the principal pathway to repair double-stranded breaks (DSBs) in vertebrate cells (29,30). Identificat...

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“…In contrast, their irradiated nontransgenic littermates developed thymic lymphoma, as evidenced by invasive thymic masses that compromised respiration. transfected with two different recombination substrate plasmids, and this bias is enforced at the joining, rather than the cleavage, step of V(D)J recombination (51). However, in thymocytes from irradiated SCID mice, the ratio of cis-to-trans rearrangements is approximately 5:1 (80), suggesting that the absence of functional DNA-PK greatly promotes interlocus misjoining events.…”

Section: Discussionmentioning

confidence: 99%

Irradiation Promotes V(D)J Joining and RAG-Dependent Neoplastic Transformation in SCID T-Cell Precursors

Williams

Grandal

Vesprini

et al. 2001

Molecular and Cellular Biology

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Defects in the nonhomologous end-joining (NHEJ) pathway of double-stranded DNA break repair severely impair V(D)J joining and selectively predispose mice to the development of lymphoid neoplasia. This connection was first noted in mice with the severe combined immune deficient (SCID) mutation in the DNA-dependent protein kinase (DNA-PK). SCID mice spontaneously develop thymic lymphoma with low incidence and long latency. However, we and others showed that low-dose irradiation of SCID mice dramatically increases the frequency and decreases the latency of thymic lymphomagenesis, but irradiation does not promote the development of other tumors. We have used this model to explore the mechanistic basis by which defects in NHEJ confer selective and profound susceptibility to lymphoid oncogenesis. Here, we show that radiation quantitatively and qualitatively improves V(D)J joining in SCID cells, in the absence of T-cell receptor-mediated cellular selection. Furthermore, we show that the lymphocyte-specific endonuclease encoded by the recombinaseactivating genes (RAG-1 and RAG-2) is required for radiation-induced thymic lymphomagenesis in SCID mice. Collectively, these data suggest that irradiation induces a DNA-PK-independent NHEJ pathway that facilitates V(D)J joining, but also promotes oncogenic misjoining of RAG-1/2-induced breaks in SCID T-cell precursors.Lymphocytes require the faithful execution of DNA repair processes to generate a highly diverse repertoire of antigen receptors. The variable region exon of each T-cell receptor (TCR) and B-cell antigen receptor (BCR) gene is assembled by site-specific cleavage and rejoining of variable (V), diversity (D), and joining (J) gene segments in developing T and B lymphocytes. Tandem genomic arrays of dozens to hundreds of V, D, or J gene segments allow combinatorial diversification of the available germline repertoire during lymphocyte development to create a large number of clonally distinct VDJ or VJ genes. In addition, terminal deoxynucleotidal transferase (TdT), a lymphocyte-specific polymerase, inserts non-germline-encoded nucleotides at V(D)J junctions, providing additional somatic diversification of the available germline repertoire (30,44,67). Thus, V(D)J recombination endows T and B lymphocytes with the capacity to specifically recognize and destroy an almost infinite array of pathogens. However, DNA breaks are highly recombinogenic and must be repaired efficiently to maintain genomic stability and minimize the risk of oncogenic transformation (31,33,43,49,90,104). A high frequency of lymphoid tumors have chromosomal translocations involving antigen receptor genes (reviewed in reference 101), raising the question as to whether the V(D)J recombination process threatens genomic stability in lymphoid lineages.The molecular mechanism of V(D)J cleavage has recently been elucidated by elegant biochemical studies (reviewed in references 93 and 106). This process is mediated by the lymphoid-specific RAG-1 and RAG-2 proteins, which bind recombination signal sequences fl...

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Intermolecular V(D)J Recombination Is Prohibited Specifically at the Joining Step

Cited by 22 publications

References 34 publications

Intermolecular V(D)J Recombination

Intermolecular V(D)J Recombination

Activation of V(D)J Recombination Induces the Formation of Interlocus Joints and Hybrid Joints in scid Pre-B-Cell Lines

Irradiation Promotes V(D)J Joining and RAG-Dependent Neoplastic Transformation in SCID T-Cell Precursors

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