Genetic interaction between the non-homologous end-joining factors during B and T lymphocyte development: In vivo mouse models

Non-homologous end joining (NHEJ) is the main DNA repair mechanism for the repair of double-strand breaks (DSBs) throughout the course of the cell cycle. DSBs are generated in developing B and T lymphocytes during V(D)J recombination to increase the repertoire of B and T cell receptors. DSBs are also generated during the class switch recombination (CSR) process in mature B lymphocytes, providing distinct effector functions of antibody heavy chain constant regions. Thus, NHEJ is important for both V(D)J recombination and CSR. NHEJ comprises core Ku70 and Ku80 subunits that form the Ku heterodimer, which binds DSBs and promotes the recruitment of accessory factors (e.g., DNA-PKcs, Artemis, PAXX, MRI) and downstream core factors (XLF, Lig4 and XRCC4). In recent decades, new NHEJ proteins have been reported, increasing complexity of this molecular pathway. Numerous in vivo mouse models have been generated and characterized to identify the interplay of NHEJ factors and their role in development of adaptive immune system. This review summarizes the currently available mouse models lacking one or several NHEJ factors, with a particular focus on early B cell development. We also underline genetic interactions and redundancy in the NHEJ pathway in mice.     

Severe combined immunodeficiency and microcephaly in siblings with hypomorphic mutations in DNA ligase IV

DNA double-strand breaks (dsb) during V(D)J recombination of T and B lymphocyte receptor genes are resolved by the non-homologous DNA end joining pathway (NHEJ) including at least six factors: Ku70, Ku80, DNA-PK(cs), Artemis, Xrcc4, and DNA ligase IV (Lig4). Artemis and Lig4 are the only known V(D)J/NHEJ factors found deficient in human genetic disorders. Null mutations of the Artemis gene result in a complete absence of T and B lymphocytes and increased cellular sensitivity to ionizing radiations, causing radiosensitive-SCID. Mutations of Lig4 are exclusively hypomorphic and have only been described in six patients, four exhibiting mild immunodeficiency associated with microcephaly and developmental delay, while two patient had leukemia. Here we report a SCID associated with microcephaly caused by compound heterozygous hypomorphic mutations in Lig4. Residual activity of Lig4 in these patients is underscored by a normal pattern of TCR-alpha and -beta junctions in the T cells of the patients and a moderate impairment of V(D)J recombination as tested in vitro. These observations contrast with the severity of the clinical immunodeficiency, suggesting that Lig4 may have additional critical roles in lymphocyte survival beyond V(D)J recombination.     

哺乳动物细胞DNA非同源末端连接及其生物学意义

DNA双链断裂是发生在哺乳动物细胞基因组水平上最严重的损伤。双链断裂得不到修复,细胞将会死亡或发生染色体断裂、丢失,若是错误修复将导致基因突变或基因组不稳定,增加癌症的风险度。哺乳动物细胞有两种重要的DNA双链断裂修复方式:非同源末端连接和同源重组。非同源末端连接途径除了在DNA双链断裂修复中起重要作用外,在V(D)J重组、HIV-1病毒整合宿主基因,以及假基因和重复序列的插入上也起着重要作用。由于非同源末端连接参与了机体许多重要的生理过程,因而其研究受到极大关注,并取得突破性的进展。     

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.     

Sibling rivalry: competition between Pol X family members in V(D)J recombination and general double strand break repair

Summary: The nonhomologous end‐joining pathway is a major means for repairing double‐strand breaks (DSBs) in all mitotic cell types. This repair pathway is also the only efficient means for resolving DSB intermediates in V(D)J recombination, a lymphocyte‐specific genome rearrangement required for assembly of antigen receptors. A role for polymerases in end‐joining has been well established. They are a major factor in determining the character of repair junctions but, in contrast to ‘core’ end‐joining factors, typically appear to have a subtle impact on the efficiency of end‐joining. Recent work implicates several members of the Pol X family in end‐joining and suggests surprising complexity in the control of how these different polymerases are employed in this pathway.     

The pathways and outcomes of mycobacterial NHEJ depend on the structure of the broken DNA ends

Mycobacteria can repair DNA double-strand breaks (DSBs) via a nonhomologous end-joining (NHEJ) system that includes a dedicated DNA ligase (LigD) and the DNA end-binding protein Ku. Here we exploit an improved plasmid-based NHEJ assay and a collection of Mycobacterium smegmatis strains bearing deletions or mutations in Ku or the DNA ligases to interrogate the contributions of LigD's three catalytic activities (polymerase, ligase, and 3' phosphoesterase) and structural domains (POL, LIG, and PE) to the efficiency and molecular outcomes of NHEJ in vivo. By analyzing in parallel the repair of blunt, 5' overhang, and 3' overhang DSBs, we discovered a novel end-joining pathway specific to breaks with 3' overhangs that is Ku- and LigD-independent and perfectly faithful. This 3' overhang NHEJ pathway is independent of ligases B and C; we surmise that it relies on NAD(+)-dependent LigA, the essential replicative ligase. We find that efficient repair of blunt and 5' overhang DSBs depends stringently on Ku and the LigD POL domain, but not on the LigD polymerase activity, which mainly serves to promote NHEJ infidelity. The lack of an effect of PE-inactivating LigD mutations on NHEJ outcomes, especially the balance between deletions and insertions at blunt or 5' overhang breaks, argues against LigD being the catalyst of deletion formation. Ligase-inactivating LigD mutations (or deletion of the LIG domain) have a modest impact on the efficiency of blunt and 5' overhang DSB repair, because the strand sealing activity can be provided in trans by one of the other resident ATP-dependent ligases (likely LigC). Reliance on the backup ligase is accompanied by a drastic loss of fidelity during blunt end and 5' overhang DSB repair. We conclude that the mechanisms of mycobacterial NHEJ are many and the outcomes depend on the initial structures of the DSBs and the available ensemble of end-processing and end-sealing components, which are not limited to Ku and LigD.     

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.     

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.     

DNA repair pathways involved in anaphase bridge formation

Cancer cells frequently exhibit gross chromosomal alterations such as translocations, deletions, or gene amplifications an important source of chromosomal instability in malignant cells. One of the better-documented examples is the formation of anaphase bridges-chromosomes pulled in opposite directions by the spindle apparatus. Anaphase bridges are associated with DNA double strand breaks (DSBs). While the majority of DSBs are repaired correctly, to restore the original chromosome structure, incorrect fusion events also occur leading to bridging. To identify the cellular repair pathways used to form these aberrant structures, we tested a requirement for either of the two major DSB repair pathways in mammalian cells: homologous recombination (HR) and nonhomologous end joining (NHEJ). Our observations show that neither pathway is essential, but NHEJ helps prevent bridges. When NHEJ is compromised, the cell appears to use HR to repair the break, resulting in increased anaphase bridge formation. Moreover, intrinsic NHEJ activity of different cell lines appears to have a positive trend with induction of bridges from DNA damage.     

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.     

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.     

Different types of V(D)J recombination and end-joining defects in DNA double-strand break repair mutant mammalian cells

The end-joining pathway of DNA double-strand break (DSB) repair is necessary for proper V(D)J recombination and repair of DSB caused by ionizing radiation. This DNA repair pathway can either use short stretches of (micro)homology near the DNA ends or use no homology at all (direct end-joining). We designed assays to determine the relative efficiencies of these (sub)pathways of DNA end-joining. In one version, a DNA substrate is linearized in such a way that joining on a particular microhomology creates a novel restriction enzyme recognition site. In the other one, the DSB is made by the RAG1 and RAG2 proteins. After PCR amplification of the junctions, the different end-joining modes can be discriminated by restriction enzyme digestion. We show that inactivation of the 'classic' end-joining factors (Ku80, DNA-PK(CS), ligase IV and XRCC4) results in a dramatic increase of microhomology-directed joining of the linear substrate, but very little decrease in overall joining efficiency. V(D)J recombination, on the other hand, is severely impaired, but also shows a dramatic shift towards microhomology use. Interestingly, two interstrand cross-linker-sensitive cell lines showed decreased microhomology-directed end-joining, but without an effect on V(D)J recombination. These results suggest that direct end-joining and microhomology-directed end-joining constitute genetically distinct DSB repair pathways.     

Modulation of X-ray-induced damage recognition and repair in ageing human peripheral blood mononuclear cells by an interleukin-6-type cytokine

We have investigated the effects of an interleukin (IL)-6-type cytokine on the DNA-binding activity of ku and on unscheduled DNA repair in X-ray-treated peripheral blood mononuclear cells (PBMC) from human subjects of different ages. The cytokine used, called K-7/D-6, is an IL-6 variant with increased in vivo and in vitro biological activity compared to the wild type molecule. Ku is the DNA-binding component of the DNA-dependent protein kinase (DNA-PK). It binds the ends of various types of DNA discontinuity and is involved in the repair of DNA breaks caused by V(D)J recombination, isotype switching, physiological oxidation reactions, ionizing radiation and some chemotherapeutic drugs. The ku-dependent repair process, called non-homologous end joining, is the main DNA double strand break repair mechanism in irradiated mammalian cells. Results show that K-7/D-6 significantly increases DNA-binding activity of ku in irradiated PBMC from young but not from elderly subjects. However, K-7/D-6 is able to induce unscheduled DNA repair in irradiated PBMC from both young and elderly subjects. These effects of K-7/D-6 are relevant to the mechanisms of the cellular response to DNA damage.     

Modulation of X-ray-induced damage recognition and repair in ageing human peripheral blood mononuclear cells by an interleukin-6-type cytokine

We have investigated the effects of an interleukin (IL)-6-type cytokine on the DNA-binding activity of ku and on unscheduled DNA repair in X-ray-treated peripheral blood mononuclear cells (PBMC) from human subjects of different ages. The cytokine used, called K-7/D-6, is an IL-6 variant with increased in vivo and in vitro biological activity compared to the wild type molecule. Ku is the DNA-binding component of the DNA-dependent protein kinase (DNA-PK). It binds the ends of various types of DNA discontinuity and is involved in the repair of DNA breaks caused by V(D)J recombination, isotype switching, physiological oxidation reactions, ionizing radiation and some chemotherapeutic drugs. The ku-dependent repair process, called non-homologous end joining, is the main DNA double strand break repair mechanism in irradiated mammalian cells. Results show that K-7/D-6 significantly increases DNA-binding activity of ku in irradiated PBMC from young but not from elderly subjects. However, K-7/D-6 is able to induce unscheduled DNA repair in irradiated PBMC from both young and elderly subjects. These effects of K-7/D-6 are relevant to the mechanisms of the cellular response to DNA damage.     

Adenovirus E4 34k and E4 11k inhibit double strand break repair and are physically associated with the cellular DNA-dependent protein kinase.

The adenovirus oncoproteins E4 34k and E4 11k, the products of E4 open reading frames 6 and 3, respectively, individually prevent the formation of concatemers of the linear viral genome in infected cells. We show here that genome concatenation in E4 mutant-infected cells requires the cellular DNA-dependent protein kinase (DNA PK) and that E4 34k inhibits V(D)J recombination, a normal cellular process that is also dependent on DNA PK. We further show that both E4 34k and E4 11k coimmunoprecipitate with DNA PK. These observations indicate that E4 products block formation of concatemers of the viral genome by inhibiting DNA PK-dependent double strand break repair and suggest that they act by forming a physical complex with DNA PK. DNA PK also participates in activation of p53 DNA-binding activity by DNA damage. By inhibiting DNA PK function, E4 products may block p53 activation in response to the products of viral DNA replication and thus provide a new mechanism to prevent apoptosis of infected cells.     

DNA polymerases in nonhomologous end joining: Are there any benefits to standing out from the crowd?

Chromosome breaks, often with damaged or missing DNA flanking the break site, are an important threat to genome stability. They are repaired in vertebrates primarily by nonhomologous end joining (NHEJ). NHEJ is unique among the major DNA repair pathways in that a continuous template cannot be used by DNA polymerases to instruct replacement of damaged or lost DNA. Nevertheless, at least 3 out of the 17 mammalian DNA polymerases are specifically employed by NHEJ. Biochemical and structural studies are further revealing how each of the polymerases employed by NHEJ possesses distinct and sophisticated means to overcome the barriers this pathway presents to polymerase activity. Still unclear, though, is how the resulting network of overlapping and nonoverlapping polymerase activities contributes to repair in cells. Environ. Mol. Mutagen., 2012. © 2012 Wiley Periodicals, Inc.     

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.     

The role of the non-homologous end-joining pathway in lymphocyte development

Summary: One of the most toxic insults a cell can incur is a disruption of its linear DNA in the form of a double‐strand break (DSB). Left unrepaired, or repaired improperly, these lesions can result in cell death or neoplastic transformation. Despite these dangers, lymphoid cells purposely introduce DSBs into their genome to maximize the diversity and effector functions of their antigen receptor genes. While the generation of breaks requires distinct lymphoid‐specific factors, their resolution requires various ubiquitously expressed DNA‐repair proteins, known collectively as the non‐homologous end‐joining pathway. In this review, we discuss the factors that constitute this pathway as well as the evidence of their involvement in two lymphoid‐specific DNA recombination events.     

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.