Signal joint formation is inhibited in murine scid preB cells and fibroblasts in substrates with homopolymeric coding ends.

During B and T lymphocyte development, immunoglobulin and T cell receptor genes are assembled from the germline V, (D) and J gene segments (Lewis, S.M., 1994. The mechanism of V(D)J joining: lessons from molecular, immunological and comparative analyses. Adv. Immunol. 56, 27-150). These DNA rearrangements, responsible for immune system diversity, are mediated by a site specific recombination machinery via recognition signal sequences (RSSs) composed of conserved heptamers and nonamers separated by spacers of 12 or 23 nucleotides (Lewis, S.M., 1994. The mechanism of V(D)J joining: lessons from molecular, immunological and comparative analyses. Adv. Immunol. 56, 27-150). Recombination occurs only between a RSS with a 12mer spacer and a RSS with a 23mer spacer (Lewis, S.M., 1994. The mechanism of V(D)J joining: lessons from molecular, immunological and comparative analyses. Adv. Immunol. 56, 27-150). RAG1 and RAG2 proteins cleave precisely at the RSS-coding sequence border leading to flush signal ends and coding ends with a hairpin structure (Eastman, M., Leu, T., Schatz, D., 1996. Initiation of V(D)J recombination in vitro obeying the 12/23 rule. Nature 380, 85-88; Roth, D.B., Menetski, J.P., Nakajima, P.B., Bosma, M.J., Gellert, M., 1992. V(D)J recombination: broken DNA molecules with covalently sealed (hairpin) coding ends in scid mouse thymocytes. Cell 983-991: Roth, D.B., Zhu, C., Gellert. M., 1993. Characterization of broken DNA molecules associated with V(D)J recombination. Proc. Natl. Acad. Sci. USA 90, 10,788-10,792; van Gent, D., McBlane, J.. Sadofsky, M., Hesse, J., Gellert, M., 1995. Initiation of V(D)J recombination in a cell-free system. Cell 81, 925-934). Signal ends join, forming a signal joint. The hairpin coding ends are opened by a yet unknown endonuclease, and are further processed to form the coding joint (Lewis, S.M., 1994. The mechanism of V(D)J joining: lessons from molecular, immunological and comparative analyses. Ad. Immunol. 56, 27-150.) The murine scid mutation has been shown to affect coding joints, but much less signal joint formation. In this study we demonstrate that the murine scid mutation inhibits correct signal joint formation when both coding ends contain homopolymeric sequences. We suggest that this finding may be due to the function of the SCID protein as an assembly component in V(D)J recombination.     

Distinct effects of DNA-PKcs and Artemis inactivation on signal joint formation in vivo

The assembly of functional immune receptor genes via V(D)J recombination in developing lymphocytes generates DNA double-stranded breaks intermediates that are repaired by non-homologous end joining (NHEJ). This repair pathway requires the sequential recruitment and activation onto coding and signal DNA ends of several proteins, including the DNA-dependent protein kinase and the nuclease Artemis. Artemis activity, triggered by the DNA-dependent protein kinase, is necessary to process the genes hairpin-sealed coding ends but appears dispensable for the ligation of the reciprocal phosphorylated, blunt-ended signal ends into a signal joint. The DNA-dependent protein kinase is however present on signal ends and could potentially recruit and activate Artemis during signal joint formation. To determine whether Artemis plays a role during the resolution of signal ends during V(D)J recombination, we analyzed the structure of signal joints generated in developing thymocytes during the rearrangement of T cell receptor genes in wild type mice and mice mutated for NHEJ factors. These joints exhibit junctional diversity resulting from N nucleotide polymerization by the terminal nucleotidyl transferase and nucleotide loss from one or both of the signal ends before they are ligated. Our results show that Artemis participates in the repair of signal ends in vivo. Furthermore, our results also show that while the DNA-dependent protein kinase complex protects signal ends from processing, including deletions, Artemis seems on the opposite to promote their accessibility to modifying enzymes. In addition, these data suggest that Artemis might be the nuclease responsible for nucleotide loss from signal ends during the repair process.     

Normal V(D)J recombination in cells from patients with Nijmegen breakage syndrome

The majority of antigen receptor diversity in mammals is generated by V(D)J recombination. During this process DNA double strand breaks are introduced at recombination signals by lymphoid specific RAG1/2 proteins generating blunt ended signal ends and hairpinned coding ends. Rejoining of all DNA ends requires ubiquitously expressed DNA repair proteins, such as Ku70/86 and DNA ligase IV/XRCC4. In addition, the formation of coding joints depends on the function of the scid gene encoding the catalytic subunit of DNA-dependent protein kinase, DNA-PK(CS), that is somehow required for processing of coding end hairpins. Recently, it was shown that purified RAG1/2 proteins can cleave DNA hairpins in vitro, but the same activity was also described for a protein complex of the DNA repair proteins Nbs1/Mre11/Rad50. This leaves the possibility that either protein complex might be involved in coding end processing in V(D)J recombination. We have therefore analyzed V(D)J recombination in cells from patients with Nijmegen breakage syndrome, carrying a mutation in the nbs1 gene. We find that V(D)J recombination frequencies and the quality of signal and coding joining are comparable to wild-type controls, as analyzed by a cellular V(D)J recombination assay. In addition, we did not detect significant differences in CDR3 sequences of endogenous Ig lambdaL and kappaL chain gene loci cloned from peripheral blood lymphocytes of an NBS patient and of healthy individuals. These findings suggest that the Nbs1/Mre11/Rad50 complex is not involved in coding end processing of V(D)J recombination.     

How to make ends meet in V(D)J recombination

In most vertebrate species analyzed so far, the diversity of soluble or membrane-bound antigen-receptors expressed by B and T lymphocytes is generated by V(D)J recombination. During this process, the coding regions for the variable domains of antigen-receptors are created by the joining of subexons that are randomly selected from arrays of tandemly repeated V, D (sometimes) and J gene segments. This involves the site-specific cleavage of chromosomal DNA by the lymphocyte-specific recombination-activating gene (RAG)-1/2 proteins, which appear to have originated from an ancient transposable element. The DNA double-strand breaks created by RAG proteins are subsequently processed and rejoined by components of the nonhomologous DNA end-joining pathway, which is conserved in all eukaryotic organisms - from unicellular yeast up to highly complex mammalian species.     

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.     

Evidence of a critical architectural function for the RAG proteins in end processing,protection, and joining in V(D)J recombination

In addition to creating the DNA double strand breaks that initiate V(D)J recombination, the RAG proteins are thought to play a critical role in the joining phase of the reaction. One such role, suggested by in vitro studies, might be to ensure the structural integrity of postcleavage complexes, but the significance of such a function in vivo is unknown. We have identified RAG1 mutants that are proficient in DNA cleavage but defective in their ability to interact with coding ends after cleavage and in the capture of target DNA for transposition. As a result, these mutants exhibit severe defects in hybrid joint formation, hairpin coding end opening, and transposition in vitro, and in V(D)J recombination in vivo. Our results suggest that the RAG proteins have an architectural function in facilitating proper and efficient V(D)J joining, and a protective function in preventing degradation of broken ends prior to joining.     

V(D)J recombination and the cell cycle

V(D)J recombination is a major source of antigen receptor diversity and represents the only known form of site-specific DNA rearrangement in vertebrates. V(D)J recombination is initiated by specific DNA cleavage at recombinational signal sequences and requires components of the general machinery used for double-strand (DS)-break repair. The involvement of DS cleavage and repair mechanisms suggests that V(D)J recombination might be coupled to the cell cycle, as introduction or persistence of DS breaks during DNA replication or mitosis could interfere with faithful transmission of genetic information to daughter cells. Here, Weei-Chin Lin and Stephen Desiderio review recent evidence indicating that this is indeed the case and consider some biological implications of this linkage.     

Coding Joint Diversity in Mature and Immature B-Cell Lines

Antigen receptor gene rearrangement is regulated by many factors in B and T lymphocytes. The sequences of the gene segments themselves, their associated recombination signal sequences (RSS), expression of the RAG genes and the chromatin accessibility of the particular gene segments to be rearranged all influence the outcome of recombination and thus antigen receptor diversity. In the present study, we have evaluated the effect of variations in RAG activity level on the junctional diversity of coding joint sequences. Using the pre-B-like 204-1-8 and the mature B DR3 cell lines under different transfection conditions, we were able to investigate recombination activity levels that varied 100-fold. We evaluated the sequences of the coding joints for junctional diversity resulting from nucleotide addition or deletion. Surprisingly, we found that the sequence of coding joints of these recombinants did not exhibit significant variation despite the large difference in recombination frequency. Our results indicate that the fidelity of the joining phase of V(D)J recombination is not jeopardized by varying RAG activity.     

Identification of an aberrant type of rearrangement in the T-cell receptor α/δ locus in adult T-cell leukemia

V(D)J recombination is the mechanism by which antigen receptor genes are assembled by three basic steps: cleavage, processing of broken DNA ends, and joining. In this process of recombination, the broken DNA molecules excised from different receptor gene loci are often joined to generate interlocus joints. The interlocus recombination process contributes to the translocation between antigen receptor genes and oncogenes, leading to the malignant transformation of lymphocytes. The α and δ chain of the T-cell receptor (TCR α/δ) locus at chromosome 14q11 is also a region where several types of chromosome translocations occur in T-cell malignancies. In the process of analyzing TCR α rearrangements in a patient with adult T-cell leukemia (ATL) carrying a translocation at chromosome 14q11, we found novel complex rearrangements in the Jα locus. On the one hand, the V2.3 gene is joined to the heptamer–nonamer recombination signal sequence of the J37 gene, and, on the other hand, the J37 gene is joined to the V2.3 recombination signal sequence through head-to-head fusion. These recombination products or hybrid joints originated through an inversion of about 70 kb DNA. Interestingly, the inverted DNA stretch contains a normal V8.1–J40 rearrangement. These findings are the first direct demonstration that successive rearrangements with hybrid joints occur on the same chromosome in the human TCR α locus. Received: August 13, 2001 / Accepted: September 25, 2001     

Thymocyte differentiation in γ-irradiated severe-combined immunodeficient mice: characterization of intermediates and products of V(D)J recombination at the T cell receptor α locus

Treatment with DNA-damaging agents promotes rescue of V(D)J recombination, limited thymocyte differentiation, and development of thymic lymphomas in severe-combined immunodeficient (SCID) mice. One intriguing aspect of this system is that irradiation rescues rearrangements at the T cell receptor (TCR) β, γ and δ loci, but not at the TCR α locus. Current models posit that only those loci that are recombinationally active at the time of irradiation can be rescued. Here, we employ sensitive, semiquantitative ligation-mediated polymerase chain reaction assays to detect a specific class of recombination intermediates, hairpin coding ends, at the TCR α locus. We found that Jα-coding ends are undetectable in unirradiated SCID thymocytes, but accumulate after irradiation at times coincident with the emergence of a CD4⁺CD8⁺ thymocyte population. Coding joints produced by joining of these ends, however, are extremely rare. To test whether the presence of hairpin coding ends at TCR α is sufficient for irradiation-mediated rescue of coding joint formation, we administered a second dose of γ-irradiation after abundant CD4⁺ CD8⁺ thymocytes and hairpin TCR α coding ends had accumulated. This treatment failed to stimulate rescue of TCR α coding joints. Thus, the presence of hairpin coding ends at the time of irradiation, while perhaps necessary, is not sufficient for rescue of V(D)J rearrangements. These results support a refined model for irradiation-mediated rescue of TCR rearrangements in SCID mice.     

The 12/23 rule is enforced at the cleavage step of V(D)J recombination in vivo

Background : V(D)J recombination is initiated by the introduction of double-stranded breaks (DSB) adjacent to recombination signal sequences (RSS). Each RSS contains a conserved heptamer and a conserved nonamer element separated by a 12 or 23 nucleotide spacer. In vivo , efficient recombination requires one RSS of each spacer length, although it has been unclear whether this '12/23 rule' regulates cleavage, joining, or both.
Results : We describe a novel system that permits semiquantitative detection of DSB at RSS derived from V(D)J recombination substrates transfected into cultured cells. This approach provides a powerful new tool for analysis of the cleavage and joining steps of V(D)J recombination in vivo . In this study, substrates containing either a consensus 12/23 RSS pair or various deviations from the consensus were used to investigate the requirements for cleavage. The results show that both a 12-spacer and a 23-spacer RSS are required for efficient cleavage. Truncated RAG-1 and RAG-2 proteins, while capable of cleaving at isolated RSS in cell-free systems, also require a 12/23 RSS pair for efficient cleavage in vivo .
Conclusions : These results suggest that the 12/23 rule is enforced at or prior to cleavage and support a synapsis model for V(D)J recombination. Detection of rare cleavage events in substrates containing a single RSS or a pair of RSS with the same spacer length provide evidence for an inefficient, single RSS cleavage pathway that may contribute to aberrant V(D)J rearrangements in vivo .     

V(D)J recombination catalyzed by mutant RAG proteins lacking consensus DNA-PK phosphorylation sites

The process of antigen receptor gene rearrangement, V(D)J recombination, involves DNA cleavage by the RAG-1 and RAG-2 proteins. Cleavage generates covalently sealed (hairpin) DNA ends, termed coding ends, which must be opened by an endonuclease prior to joining. Resolution of these hairpin ends requires the activity of the DNA-dependent protein kinase (DNA-PK), a protein kinase whose specific role is yet undetermined. It has been suggested that phosphorylation of one or both RAG proteins by DNA-PK is required to activate or recruit the hairpin-opening nuclease. Furthermore, very recent work has shown that RAG proteins themselves can open hairpins. These data raise the possibility that DNA-PK-mediated phosphorylation of the RAG proteins could regulate the hairpin opening reaction. To test this hypothesis, we constructed mutant versions of RAG-1 and RAG-2 in which all four DNA-PK consensus phosphorylation sites were removed by site-directed mutagenesis. Our data provide conclusive evidence that phosphorylation of these conserved serine/threonine residues is not required for hairpin opening or joining of V(D)J recombination intermediates.     

Ongoing V kappa-J kappa recombination after formation of a productive V kappa-J kappa coding joint.

V kappa genes of man can recombine with the J kappa gene segments either by an inversion or by a deletion mechanism. Back-to-back fusion products of the respective recombination signal sequences (signal joints) are retained on the chromosome after the formation of a V kappa-J kappa coding joint by an inversion. Our knowledge of the structure of the human kappa locus and the application of the polymerase chain reaction allowed us now to establish a direct relationship between different kappa recombination products in the lymphoid cell line JI. Two consecutive inversions fully explain the existence of two coding joints and two signal joints on the same chromosome of this cell line. Although the initially formed coding joint is productively rearranged and expressed, a second V kappa-J kappa rearrangement took place which leads to an aberrant joint. In this process a J kappa gene segment of the signal joint that had been created in the first V kappa-J kappa joining was used as the recombination target. The sequence of the two rearrangements is unequivocal since a product of the first (productive) reaction is a partner in the second (aberrant) one.     

The role of recombination signal sequences in the preferential joining by deletion in DH-JH recombination and in the ordered rearrangement of the IgH locus

The bias favoring deletion over inversion in DH-JH rearrangement has been known for years, but the underlying mechanism has yet to be fully defined. It has been suggested that the ratio of deletion/inversion is determined by the combined effect of two factors: (i) the relative strengths of 5' and 3' recombination signal sequences (RSS) of a DH segment, and (ii) the efficiency with which the deletional product (one joint) forms relative to the inversional product (two joints). In this study, we analyzed for the first time the effect of factor 1 alone on the biased 3' RSS utilization in DH-JH joining by using deletional plasmids in an extrachromosomal substrate V(D)J recombination assay. It was found that the 3' RSS and associated coding end (12 bp) mediate recombination more efficiently than the 5' RSS/coding end DH-JH plasmids. These results demonstrate that the effect of the RSS/coding end alone can account, at least partially, for the predominant deletion in DH-JH recombination. The potential effect of the relative strength of RSS and associated coding end on the ordered rearrangement of DH-JH followed by VH to DH-JH was also assessed. When recombination frequencies of D-->J (3' DH to J3) were compared with frequencies of V-- >D (VHPJ14 to 3' DH or VHOX2 to 3' DH), it was found that V-->D joining was, if anything, more efficient than D-->J joining. Therefore, if all three segments were accessible, RSS/coding end effects would not contribute to the ordered rearrangement of the IgH locus.      

Artemis sheds new light on V(D)J recombination

Summary: V(D)J recombination represents one of the three mechanisms that contribute to the diversity of the immune repertoire of B lymphocytes and T lymphocytes. It also constitutes a major checkpoint during the development of the immune system. Indeed, any V(D)J recombination deficiency leads to a block of B‐cell and T‐cell maturation in humans and animal models, leading to severe combined immunodeficiency (T‐B‐SCID). Nine factors have been identified so far to participate in V(D)J recombination. The discovery of Artemis, mutated in a subset of T‐B‐SCID, provided some new information regarding one of the missing V(D)J recombinase activities: hairpin opening at coding ends prior to DNA repair of the recombination activating genes 1/2‐generated DNA double‐strand break. New conditions of immune deficiency in humans are now under investigations and should lead to the identification of additional V(D)J recombination/DNA repair factors.     

Structure of extrachromosomal circular DNAs generated by immunoglobulin light chain gene rearrangements

Recombination at the immunoglobulin kappa or lambda light chain locus generates extrachromosomal circular DNAs. We have isolated circular DNAs from adult mouse spleen cells and prepared a circular DNA clone library. We characterized four J kappa-positive and one J lambda 1-positive clones. The J kappa-clones contained both coding and signal joints of V kappa-J kappa joining, and the J lambda 1-clone contained a signal joint of V lambda 1-J lambda 1 joining. Genomic organization of the V kappa gene families used in these joints suggested the excision of circular DNA preceded by inversion. A specific dinucleotide (P) insertion in the coding joint was observed in two clones. Three coding joints were out of frame and one clone had an in-frame coding joint, although possibly combined with a pseudo-V kappa gene. These kappa-positive circular DNAs are possibly excised from the chromosome by secondary recombinations which replace non-productive primary rearrangements.     

A new gene involved in DNA double-strand break repair and V(D)J recombination is located on human chromosome 10p

V(D)J recombination, accountable for the diversity of T cell receptor- and immunoglobulin-encoding genes, is initiated by a lymphoid-specific DNA double-strand break. The general DNA repair machinery is responsible for the resolution of this break. Any defect in one of the known components of the DNA repair/V(D)J recombination machinery (Ku70, Ku80, DNA-PKcs, XRCC4 and DNA ligase IV) leads to abortion of the V(D)J rearrangement process, early block in both T and B cell maturation, and ultimately to severe combined immune deficiency (SCID) in several animal models. A human SCID condition is also characterized by an absence of mature T and B lymphocytes, and is associated with an increase in sensitivity to DNA-damaging agents (RS-SCID). None of the above-mentioned genes are defective in these patients, arguing for the likelihood of the existence of yet another unknown component of the V(D)J recombination/DNA repair apparatus. Athabascan-speaking (SCIDA) Navajo and Apache Native Americans have a very high incidence of T(-)B(-)SCID. The SCIDA locus is highly linked with markers on chromosome 10p, although the exact molecular defect has not been recognized in these patients. We show here that cells with the SCIDA defect are impaired in the DNA repair phase of V(D)J recombination similarly to RS-SCID, precisely an absence of V(D)J coding joint formation. Moreover, genotyping analysis in several RS-SCID families corroborates a linkage of the RS-SCID locus to the SCIDA region on chromosome 10p. These results demonstrate the presence of a new essential DNA repair/V(D)J recombination gene in this region, the mutation of which causes RS-SCID in humans.     

The basis for the mechanistic bias for deletional over inversional V(D)J recombination.

V(D)J recombination between recognition sites in the genome is characterized by certain biases. At some loci, proximal sites undergo recombination substantially more frequently than distal ones. The joining of DH/JH is an example of this. Because the DH element bears signal sequences on each side, inversion would be expected as often as deletion in DH/JH recombination. However, the markedly favored outcome is deletion, entailing utilization of the closer recombination site. One model proposed to explain these biases is the tracking model in which the recombinase tracks from one site to the other. Here, we have directly tested for various types of tracking in V(D)J recombination and have found no indication that it occurs. In addition, we have created DH-JH minilocus substrates for analysis of the basis for the preference for deletion. We find that we can reproduce the deletional bias for the system. Moreover, by flipping the orientation of the D segment, we can reverse the bias such that the frequency of inversions can exceed the number of deletions. These results indicate (1) that there is no intrinsic topological preference in this reaction, and (2) that the sequence of the signal and coding ends determines the bias.     

Recombination signal sequence variations and the mechanism of patterned T-cell receptor-beta locus rearrangement

Rearrangement of antigen receptor genes is controlled at multiple levels. One important regulation is achieved through variation in the recombination signals (RS) that flank the rearranging variable (V), diversity (D), and joining (J) gene segments. Several functional and biochemical studies have confirmed the importance of RS variations in gene rearrangement but very few molecular analyses has been reported with known, endogenous RS motifs. We have shown previously that rearrangement of the murine T-cell receptor (TCR)-B, D, and J genes follows a stereotypical pattern determined largely by the BJ genes and their flanking RS. Therefore, we studied the mechanism by which the endogenous BJ RS determine patterned gene rearrangement. We have compared the activity of three representative RS in transfection and in vitro DNA cleavage assays. Mutagenesis studies identified residues in various components of the RS and flanking coding ends that cooperate to determine the ultimate efficiency of recombination. Surprisingly, we find that changes in one component of the RS can be compensated by other elements to restore functional activity. DNA electrophoretic mobility shift assays (EMSA) show that a combinatorial effect of various higher order protein/RS complexes can, in part, control the efficiency of recombination. We propose that evolution of a patterned primary antigen receptor repertoire reflects the delicate interplay between various components of the RS and flanking coding end motifs resulting from the highly flexible interactions of the recombinase with its target DNA.