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.     

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.     

Induction of V(D)J-mediated recombination of an extrachromosomal substrate following exposure to DNA-damaging agents

V(D)J recombinase normally mediates recombination signal sequence (RSS) directed rearrangements of variable (V), diversity (D), and joining (J) germline gene segments that lead to the generation of diversified T cell receptor or immunoglobulin proteins in lymphoid cells. Of significant clinical importance is that V(D)J-recombinase-mediated rearrangements at immune RSS and nonimmune cryptic RSS (cRSS) have been implicated in the genomic alterations observed in lymphoid malignancies. There is growing evidence that exposure to DNA-damaging agents can increase the frequency of V(D)J-recombinase-mediated rearrangements in vivo in humans. In this study, we investigated the frequency of V(D)J-recombinase-mediated rearrangements of an extrachromosomal V(D)J plasmid substrate following exposure to alkylating agents and ionizing radiation. We observed significant dose- and time-dependent increases in V(D)J recombination frequency (V(D)J RF) following exposure to ethyl methanesulfonate (EMS) and methyl methanesulfonate (MMS) but not a nonreactive analogue, methylsulfone (MeSulf). We also observed a dose-dependent increase in V(D)J RF when cells were exposed to gamma radiation. The induction of V(D)J rearrangements following exposure to DNA-damaging agents was not associated with an increase in the expression of RAG 1/2 mRNA compared to unexposed controls or an increase in expression of the DNA repair Ku70, Ku80 or Artemis proteins of the nonhomologous end joining pathway. These studies demonstrate that genotoxic alkylating agents and ionizing radiation can induce V(D)J rearrangements through a cellular response that appears to be independent of differential expression of proteins involved with V(D)J recombination.     

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.     

RAG1 and RAG2 in V(D)J recombination and transposition

RAG1 and RAG2 are the key components of the V(D)J recombinase machinery that catalyses the somatic gene rearrangements of antigen receptor genes during lymphocyte development. In the first step of V(D)J recombination--DNA cleavage--the RAG proteins act together as an endonuclease to excise the DNA between two individual gene segments. They are also thought to be involved in the subsequent DNA joining step. In vitro, the RAG proteins catalyze the integration of the excised DNA element into target DNA completing a process similar to bacterial transposition. In vivo, this reaction is suppressed by an unknown mechanism. The individual roles of RAG1 and RAG2 in V(D)J recombination and transposition reactions are discussed based on mutation analyses and structure predictions.     

A hypomorphic Artemis human disease allele causes aberrant chromosomal rearrangements and tumorigenesis

The Artemis gene encodes a DNA nuclease that plays important roles in non-homologous end-joining (NHEJ), a major double-strand break (DSB) repair pathway in mammalian cells. NHEJ factors repair general DSBs as well as programmed breaks generated during the lymphoid-specific DNA rearrangement, V(D)J recombination, which is required for lymphocyte development. Mutations that inactivate Artemis cause a human severe combined immunodeficiency syndrome associated with cellular radiosensitivity. In contrast, hypomorphic Artemis mutations result in combined immunodeficiency syndromes of varying severity, but, in addition, are hypothesized to predispose to lymphoid malignancy. To elucidate the distinct molecular defects caused by hypomorphic compared with inactivating Artemis mutations, we examined tumor predisposition in a mouse model harboring a targeted partial loss-of-function disease allele. We find that, in contrast to Artemis nullizygosity, the hypomorphic mutation leads to increased aberrant intra- and interchromosomal V(D)J joining events. We also observe that dysfunctional Artemis activity combined with p53 inactivation predominantly predisposes to thymic lymphomas harboring clonal translocations distinct from those observed in Artemis nullizygosity. Thus, the Artemis hypomorphic allele results in unique molecular defects, tumor spectrum and oncogenic chromosomal rearrangements. Our findings have significant implications for disease outcomes and treatment of patients with different Artemis mutations.     

A novel missense RAG-1 mutation results in T-B-NK+ SCID in Athabascan-speaking Dine Indians from the Canadian Northwest Territories

DNA double-strand repair factors in the non-homologous end joining (NHEJ) pathway resolve DNA double-strand breaks introduced by the recombination-activating gene (RAG) proteins during V(D)J recombination of T and B lymphocyte receptor genes. Defective NHEJ and subsequent failure of V(D)J recombination leads to severe combined immunodeficiency disease (SCID). We originally linked T(-)B(-)NK(+) SCID in Athabascan-speaking Native Americans in the Southwestern US and Northwest Territories of Canada to chromosome 10. However, despite a common ancestry, the null mutation in the Artemis gene that we found to be causal in the SCID among the Navajo and Apache Indians was not present in the Dine Indians in the Northwest Territories. We now report a novel homozygous missense mutation (R776W) in RAG-1 in three children with T(-)B(-)NK(+) SCID from two related families of Athabascan-speaking Dine Indians in the Canadian Northwest Territories. As expected, we found no increased sensitivity to ionizing radiation in patient fibroblasts. The impaired activity of this RAG-1 mutant in V(D)J recombination was confirmed by the EGFP-based V(D)J recombination assays. Overexpression of wild type RAG-1 in patient fibroblasts complemented V(D)J recombination, with recovery of both coding and signal joint formation. Our results indicate that the novel R776W missense mutation in RAG-1 is causal in the T(-)B(-)NK(+) SCID phenotype in Athabascan-speaking Dine Indians from the Canadian Northwest Territories.     

The bounty of RAGs: recombination signal complexes and reaction outcomes

Summary: V(D)J recombination is a form of site‐specific DNA rearrangement through which antigen receptor genes are assembled. This process involves the breakage and reunion of DNA mediated by two lymphoid cell‐specific proteins, recombination activating genes RAG‐1 and RAG‐2, and ubiquitously expressed architectural DNA‐binding proteins and DNA‐repair factors. Here I review the progress toward understanding the composition, assembly, organization, and activity of the protein‐DNA complexes that support the initiation of V(D)J recombination, as well as the molecular basis for the sequence‐specific recognition of recombination signal sequences (RSSs) that are the targets of the RAG proteins. Parallels are drawn between V(D)J recombination and Tn5/Tn10 transposition with respect to the reactions, the proteins, and the protein‐DNA complexes involved in these processes. I also consider the relative roles of the different sequence elements within the RSS in recognition, cleavage, and post‐cleavage events. Finally, I discuss alternative DNA transactions mediated by the V(D)J recombinase, the protein‐DNA complexes that support them, and factors and forces that control them.     

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.     

Role of recombination activating genes in the generation of antigen receptor diversity and beyond

V(D)J recombination is the process by which antibody and T‐cell receptor diversity is attained. During this process, antigen receptor gene segments are cleaved and rejoined by non‐homologous DNA end joining for the generation of combinatorial diversity. The major players of the initial process of cleavage are the proteins known as RAG1 (recombination activating gene 1) and RAG2. In this review, we discuss the physiological function of RAGs as a sequence‐specific nuclease and its pathological role as a structure‐specific nuclease. The first part of the review discusses the basic mechanism of V(D)J recombination, and the last part focuses on how the RAG complex functions as a sequence‐specific and structure‐specific nuclease. It also deals with the off‐target cleavage of RAGs and its implications in genomic instability.     

Structure and function of mammalian DNA ligases

DNA joining events are required for the completion of DNA replication, DNA excision repair and genetic recombination. Five DNA ligase activities, I–V, have been purified from mammalian cell extracts and three mammalian LIG genes, LIG1, LIG3 and LIG4, have been cloned. During DNA replication, the joining of Okazaki fragments by the LIG1 gene product appears to be mediated by an interaction with proliferating cell nuclear antigen (PCNA). This interaction may also occur during the completion of mismatch, nucleotide excision and base excision repair (BER). In addition, DNA ligase I participates in a second BER pathway that is carried out by a multiprotein complex in which DNA ligase I interacts directly with DNA polymerase β. DNA ligase IIIα and DNA ligase IIIβ, which are generated by alternative splicing of the LIG3 gene, can be distinguished by their ability to bind to the DNA repair protein, XRCC1. The interaction between DNA ligase IIIα and XRCC1, which occurs through BRCT motifs in the C-termini of these polypeptides, implicates this isoform of DNA ligase III in the repair of DNA single-strand breaks and BER. DNA ligase II appears to be a proteolytic fragment of DNA ligase IIIα. The restricted expression of DNA ligase IIIβ suggests that this enzyme may function in the completion of meiotic recombination or in a postmeiosis DNA repair pathway. Complex formation between DNA ligase IV and the DNA repair protein XRCC4 involves the C-terminal region of DNA ligase IV, which contains two BRCT motifs. This interaction, which stimulates DNA joining activity, implies that DNA ligase IV functions in V(D)J recombination and non-homologous end-joining of DNA double-strand breaks. At the present time, it is not known whether DNA ligase V is derived from one of the known mammalian LIG genes or is the product of a novel gene.     

Extrachromosomal recombination substrates recapitulate beyond 12/23 restricted VDJ recombination in nonlymphoid cells

V(D)J recombination occurs efficiently only between gene segments flanked by recombination signals (RSs) containing 12 and 23 base pair spacers (the 12/23 rule). A further limitation "beyond the 12/23 rule" (B12/23) exists at the TCRbeta locus and ensures Dbeta usage. Herein, we show that extrachromosomal V(D)J recombination substrates recapitulate B12/23 restriction in nonlymphoid cells. We further demonstrate that the Vbeta coding flank, the 12-RS heptamer/nonamer, and the 23-RS spacer each can significantly influence B12/23 restriction. Finally, purified core RAG1 and RAG2 proteins (together with HMG2) also reproduce B12/23 restriction in a cell-free system. Our findings indicate that B12/23 restriction of V(D)J recombination is cemented at the level of interactions between the RAG proteins and TCRbeta RS sequences.     

Sensing of intermediates in V(D)J recombination by ATM.

Ataxia-telangiectasia mutated (ATM) is required for resistance to radiation-induced DNA breaks. Here we use chromatin immunoprecipitation to show that ATM also localizes to breaks associated with V(D)J recombination. ATM recruitment to the recombining locus correlates approximately with recruitment of the break-initiating factor RAG1 and precedes efficient break repair, consistent with localization of ATM to normal recombination intermediates. A product of ATM kinase activity, Ser 18-phosphorylated p53, was detected similarly at these breaks, arguing that ATM phosphorylates target proteins in situ. We suggest routine surveillance of intermediates in V(D)J recombination by ATM helps suppress potentially oncogenic translocations when repair fails.     

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 .     

Single-strand recombination signal sequence nicks in vivo: evidence for a capture model of synapsis

Variable (diversity) joining (V(D)J) recombination is initiated by the introduction of single-strand DNA breaks (nicks) at recombination signal sequences (RSSs). The importance and fate of these RSS nicks for the regulation of the V(D)J rearrangement and their potential contribution to genomic instability are poorly understood. Using two new methodologies, we were able to detect and quantify specific RSS nicks introduced into genomic DNA by incubation with recombination-activating gene proteins in vitro. In vivo, however, we found that nicks mediated by recombination-activating gene (RAG) proteins were detectable only in gene segments associated with RSSs containing 12-base pair spacers but not in those containing 23-base pair spacers. These data support a model of capture rather than synapsis for pairwise RSS cleavage during V(D)J recombination.     

Cyclin-dependent kinase-dependent phosphorylation of Lif1 and Sae2 controls imprecise nonhomologous end joining accompanied by double-strand break resection

DNA double-strand breaks (DSBs) are repaired by two distinct pathways, homologous recombination (HR) and nonhomologous end joining (NHEJ). NHEJ includes two pathways, that is, precise and imprecise end joining. We found that Lif1, a component of the DNA ligase IV complex in Saccharomyces cerevisiae, was phosphorylated by cyclin-dependent kinase (CDK) at Ser261 during the S to G2 phase but not during G1 phase. This phosphorylation was required for efficient NHEJ in G2/M cells, rather than in G1 cells. It also promotes the stable binding of Lif1 protein to DSBs, specifically in G2/M-arrested cells, which shows the resection of DSB ends. Thus, Lif1 phosphorylation plays a critical role in a certain type of imprecise NHEJ accompanied by DSB end resection and micro-homology. Lif1 phosphorylation at Ser261 is probably involved in micro-homology-dependent end joining associated with producing single-stranded DSB ends that are formed by Sae2 as early intermediates in the HR pathway. CDK-dependent modification of the NHEJ pathway might make DSB ends compatible for NHEJ and thus prevent competition between HR and NHEJ in hierarchy on the choice of DSB repair pathways.     

Regions of RAG1 protein critical for V(D)J recombination

The products of the recombination activating genes RAG1 and RAG2 are essential for activating V(D)J recombination, and thus are indispensable for the production of functional and diverse antigen receptors. To investigate the function of RAG1, we have tested a series of insertion and substitution mutations for their ability to induce V(D)J rearrangement on both deletional and inversional plasmid substrates. With these substrates we were also able to assess the effects of these mutations on both coding and signal joint formation, and to show that any one mutant affected all these reactions similarly. As defined previously, the core active regions of RAG1 and RAG2 permit the deletion of 40% and 25%, respectively, of well-conserved sequence. We show here that this “dispensable” region of RAG1 is not necessary for coding joint formation or for recombination of an integrated substrate, and that this portion is not functionally redundant with the “dispensable” region of RAG2. Recombination with these core regions is also still subject to the 12/23 joining rule. Further, the minimal essential core region of RAG1 can be located within an even smaller portion of the gene.     

Recombinational DNA Repair in Cancer and Normal Cells: The Challenge of Functional Analysis

A major goal of current cancer research is to understand the functional consequences of mutations in recombinational DNA repair genes. The introduction of artificial recombination substrates into living cells has evolved into a powerful tool to perform functional analysis of DNA double strand break (DSB) repair. Here, we review the principles and practice of current plasmid assays with regard to the two major DSB repair pathways, homologous recombination and nonhomologous end-joining. A spectrum of assay types is available to assess repair in a wide variety of cell lines. However, several technical challenges still need to be overcome. Understanding the alterations of DSB repair in cancers will ultimately provide a rational basis for drug design that may selectively sensitize tumor cells to ionizing radiation and chemotherapy, thereby achieving therapeutic gain.     

The sticky business of histone H2AX in V(D)J recombination,maintenance of genomic stability,and suppression of lymphoma

DNA double strand breaks (DSBs) induced during cellular metabolism, DNA replication, and genomic rearrangement events lead to phosphorylation of the H2AX core histone variant in surrounding chromatin. H2AX is essential for normal DSB repair, maintenance of genomic stability, and suppression of lymphomas with clonal translocations and intra-chromosomal deletions. One current focus of our lab is to elucidate mechanisms through which H2AX functions in the cellular DNA damage response using V(D)J recombination as a model system. A number of potential H2AX functions can be readily tested using novel experimental approaches developed in our lab. These putative functions include: (1) modulation of chromatin accessibility to facilitate kinetics of DSB repair, (2) stabilization of broken DNA strands to maintain ends in close proximity, and (3) amplification of DNA damage signals. Here, we summarize our recent efforts in elucidating mechanisms by which H2AX functions during V(D)J recombination to coordinate DSB repair with cellular proliferation and survival to prevent translocations and suppress lymphomagenesis.