Reprint of “The clinical impact of deficiency in DNA non-homologous end-joining”

DNA non-homologous end-joining (NHEJ) is the major DNA double strand break (DSB) repair pathway in mammalian cells. Defects in NHEJ proteins confer marked radiosensitivity in cell lines and mice models, since radiation potently induces DSBs. The process of V(D)J recombination functions during the development of the immune response, and involves the introduction and rejoining of programmed DSBs to generate an array of diverse T and B cells. NHEJ rejoins these programmed DSBs. Consequently, NHEJ deficiency confers (severe) combined immunodeficiency – (S)CID – due to a failure to carry out V(D)J recombination efficiently. NHEJ also functions in class switch recombination, another step enhancing T and B cell diversity. Prompted by these findings, a search for radiosensitivity amongst (S)CID patients revealed a radiosensitive sub-class, defined as RS-SCID. Mutations in NHEJ genes, defining human syndromes deficient in DNA ligase IV (LIG4 Syndrome), XLF-Cernunnos, Artemis or DNA-PKcs, have been identified in such patients. Mutations in XRCC4 or Ku70,80 in patients have not been identified. RS-SCID patients frequently display additional characteristics including microcephaly, dysmorphic facial features and growth delay. Here, we overview the clinical spectrum of RS-SCID patients and discuss our current understanding of the underlying biology.     

A novel radiosensitive SCID patient with a pronounced G2/M sensitivity

V(D)J rearrangement in lymphoid cells involves repair of double-strand breaks (DSBs) through non-homologous end joining (NHEJ). Defects in this process lead to increased radiosensitivity and severe combined immunodeficiency (RS-SCID). Here, a SCID patient, M3, is described with a T^?B⁺NK⁺ phenotype but without causative mutations in CD3δ, ?, ζ or IL7Rα, genes specifically involved in T cell development. Clonogenic survival of M3 fibroblasts showed an increased sensitivity to the DSB-inducing agents ionizing radiation and bleomycin, as well as the crosslinking compound, mitomycin C. We did not observe inactivating mutations in known NHEJ genes and results of various DSB-repair assays in G₁ M3 cells were indistinguishable from those obtained with normal cells. However, we found increased chromosomal radiosensitivity at the G₂ phase of the cell cycle. Checkpoint analysis indicated functional G₁/S and intra-S checkpoints after irradiation but impaired activation of the “early” G₂/M checkpoint. Together these results indicate a novel class of RS-SCID patients characterized by the specific absence of T lymphocytes and associated with defects in G₂-specific DSB repair. The pronounced G₂/M radiosensitivity of the RS-SCID patient described here, suggests a defect in a putative novel and uncharacterized factor involved in cellular DNA damage responses and T cell development.     

Artemis, a novel DNA double-strand break repair/V(D)J recombination protein, is mutated in human severe combined immune deficiency

The V(D)J recombination process insures the somatic diversification of immunoglobulin and antigen T cell receptor encoding genes. This reaction is initiated by a DNA double-strand break (dsb), which is resolved by the ubiquitously expressed DNA repair machinery. Human T-B-severe combined immunodeficiency associated with increased cellular radiosensitivity (RS-SCID) is characterized by a defect in the V(D)J recombination leading to an early arrest of both B and T cell maturation. We previously mapped the disease-related locus to the short arm of chromosome 10. We herein describe the cloning of the gene encoding a novel protein involved in V(D)J recombination/DNA repair, Artemis, whose mutations cause human RS-SCID. Protein sequence analysis strongly suggests that Artemis belongs to the metallo-beta-lactamase superfamily.     

Reprint of “Functional overlaps between XLF and the ATM-dependent DNA double strand break response”

Developing B and T lymphocytes generate programmed DNA double strand breaks (DSBs) during the V(D)J recombination process that assembles exons that encode the antigen-binding variable regions of antibodies. In addition, mature B lymphocytes generate programmed DSBs during the immunoglobulin heavy chain (IgH) class switch recombination (CSR) process that allows expression of different antibody heavy chain constant regions that provide different effector functions. During both V(D)J recombination and CSR, DSB intermediates are sensed by the ATM-dependent DSB response (DSBR) pathway, which also contributes to their joining via classical non-homologous end-joining (C-NHEJ). The precise nature of the interplay between the DSBR and C-NHEJ pathways in the context of DSB repair via C-NHEJ remains under investigation. Recent studies have shown that the XLF C-NHEJ factor has functional redundancy with several members of the ATM-dependent DSBR pathway in C-NHEJ, highlighting unappreciated major roles for both XLF as well as the DSBR in V(D)J recombination, CSR and C-NHEJ in general. In this review, we discuss current knowledge of the mechanisms that contribute to the repair of DSBs generated during B lymphocyte development and activation with a focus on potential functionally redundant roles of XLF and ATM-dependent DSBR factors.     

Cernunnos, a novel nonhomologous end-joining factor, is mutated in human immunodeficiency with microcephaly

DNA double-strand breaks (DSBs) occur at random upon genotoxic stresses and represent obligatory intermediates during physiological DNA rearrangement events such as the V(D)J recombination in the immune system. DSBs, which are among the most toxic DNA lesions, are preferentially repaired by the nonhomologous end-joining (NHEJ) pathway in higher eukaryotes. Failure to properly repair DSBs results in genetic instability, developmental delay, and various forms of immunodeficiency. Here we describe five patients with growth retardation, microcephaly, and immunodeficiency characterized by a profound T+B lymphocytopenia. An increased cellular sensitivity to ionizing radiation, a defective V(D)J recombination, and an impaired DNA-end ligation process both in vivo and in vitro are indicative of a general DNA repair defect in these patients. All five patients carry mutations in the Cernunnos gene, which was identified through cDNA functional complementation cloning. Cernunnos/XLF represents a novel DNA repair factor essential for the NHEJ pathway.     

Lack of MSH2 involvement differentiates V(D)J recombination from other non-homologous end joining events

V(D)J recombination and class switch recombination are the two DNA rearrangement events used to diversify the mouse and human antibody repertoires. While their double strand breaks (DSBs) are initiated by different mechanisms, both processes use non-homologous end joining (NHEJ) in the repair phase. DNA mismatch repair elements (MSH2/MSH6) have been implicated in the repair of class switch junctions as well as other DNA DSBs that proceed through NHEJ. MSH2 has also been implicated in the regulation of factors such as ATM and the MRN (Mre11, Rad50, Nbs1) complex, which are involved in V(D)J recombination. These findings led us to examine the role of MSH2 in V(D)J repair. Using MSH2-/- and MSH2+/+ mice and cell lines, we show here that all pathways involving MSH2 are dispensable for the generation of an intact pre-immune repertoire by V(D)J recombination. In contrast to switch junctions and other DSBs, the usage of terminal homology in V(D)J junctions is not influenced by MSH2. Thus, whether the repair complex for V(D)J recombination is of a canonical NHEJ type or a separate microhomology-mediated-end joining (MMEJ) type, it does not involve MSH2. This highlights a distinction between the repair of V(D)J recombination and other NHEJ reactions.     

Impaired Replication Stress Response in Cells from Immunodeficiency Patients Carrying Cernunnos/XLF Mutations

Non-Homologous End Joining (NHEJ) is one of the two major pathways of DNA Double Strand Breaks (DSBs) repair. Mutations in human NHEJ genes can lead to immunodeficiency due to its role in V(D)J recombination in the immune system. In addition, most patients carrying mutations in NHEJ genes display developmental anomalies which are likely the result of a general defect in repair of endogenously induced DSBs such as those arising during normal DNA replication. Cernunnos/XLF is a recently identified NHEJ gene which is mutated in immunodeficiency with microcephaly patients. Here we aimed to investigate whether Cernunnos/XLF mutations disrupt the ability of patient cells to respond to replication stress conditions. Our results demonstrate that Cernunnos/XLF mutated cells and cells downregulated for Cernunnos/XLF have increased sensitivity to conditions which perturb DNA replication. In addition, under replication stress, these cells exhibit impaired DSB repair and increased accumulation of cells in G2/M. Moreover Cernunnos/XLF mutated and down regulated cells display greater chromosomal instability, particularly at fragile sites, under replication stress conditions. These results provide evidence for the role of Cernunnos/XLF in repair of DSBs and maintenance of genomic stability under replication stress conditions. This is the first study of a NHEJ syndrome showing association with impaired cellular response to replication stress conditions. These findings may be related to the clinical features in these patients which are not due to the V(D)J recombination defect. Additionally, in light of the emerging important role of replication stress in the early stages of cancer development, our findings may provide a mechanism for the role of NHEJ in preventing tumorigenesis.     

Cernunnos/XLF: a new player in DNA double-strand break repair

Non-homologous end-joining (NHEJ) is the predominant repair pathway for DNA double-strand breaks (DSBs) in vertebrates and also plays a crucial role in V(D)J recombination of immunoglobulin genes. Cernunnos/XLF is a newly identified core factor for NHEJ, and its defect causes a genetic disease characterized by neural disorders, immunodeficiency and increased radiosensitivity. Cernunnos/XLF has at least two distinct functions in NHEJ. Cernunnos/XLF interacts with and stimulates the XRCC4/DNA ligase IV complex, which acts at the final ligation step in NHEJ. In living cells, Cernunnos/XLF quickly responds to DSB induction and accumulates at damaged sites in a Ku-dependent but XRCC4-independent manner. These observations indicate that Cernunnos/XLF plays a unique role in bridging damage sensing and DSB rejoining steps of NHEJ. Recent crystallographic analyses of the homodimeric Cernunnos/XLF protein provide structural insights into the Cernunnos/XLF functions. These studies offer important clues toward understanding the molecular mechanism for NHEJ-defective diseases.     

Harmonising the response to DSBs: a new string in the ATM bow

Ataxia telangiestasia mutated protein (ATM) is the major kinase that initiates the DNA damage signal transduction response following exposure to ionising radiation (IR) in mammalian cells. DNA non-homologous end-joining (NHEJ) is the most significant double strand break (DSB) repair pathway in mammalian cells. ATM-defective cell lines display cell cycle checkpoint defects and show pronounced radiosensitivity. ATM signalling was previously thought to be dispensable for NHEJ. This review discusses recent findings that ATM activates an end-processing mechanism dependent upon Artemis, a nuclease that also functions to cleave the hairpin intermediate generated during V(D)J recombination. ATM/Artemis-dependent end-processing is required for the repair of a sub-fraction (approximately 10%) of DSBs induced by IR and makes a significant contribution to survival following exposure to ionising radiation. This result represents a new role for ATM and demonstrates a novel cross communication between the DNA repair and signal transduction machinery.     

Non-homologous DNA end joining

DNA double-strand breaks (DSBs) are a serious threat for the cell and when not repaired or misrepaired can result in mutations or chromosome rearrangements and eventually in cell death. Therefore, cells have evolved a number of pathways to deal with DSB including homologous recombination (HR), single-strand annealing (SSA) and non-homologous end joining (NHEJ). In mammals DSBs are primarily repaired by NHEJ and HR, while HR repair dominates in yeast, but this depends also on the phase of the cell cycle. NHEJ functions in all kinds of cells, from bacteria to man, and depends on the structure of DSB termini. In this process two DNA ends are joined directly, usually with no sequence homology, although in the case of same polarity of the single stranded overhangs in DSBs, regions of microhomology are utilized. The usage of microhomology is common in DNA end-joining of physiological DSBs, such as at the coding ends in V(D)J (variable(diversity) joining) recombination. The main components of the NHEJ system in eukaryotes are the catalytic subunit of DNA protein kinase (DNA-PK(cs)), which is recruited by DNA Ku protein, a heterodimer of Ku70 and Ku80, as well as XRCC4 protein and DNA ligase IV. A complex of Rad50/Mre11/Xrs2, a family of Sir proteins and probably other yet unidentified proteins can be also involved in this process. NHEJ and HR may play overlapping roles in the repair of DSBs produced in the S phase of the cell cycle or at replication forks. Aside from DNA repair, NHEJ may play a role in many different processes, including the maintenance of telomeres and integration of HIV-1 genome into a host genome, as well as the insertion of pseudogenes and repetitive sequences into the genome of mammalian cells. Inhibition of NHEJ can be exploited in cancer therapy in radio-sensitizing cancer cells. Identification of all key players and fundamental mechanisms underlying NHEJ still requires further research.     

The endless tale of non-homologous end-joining

DNA double-strand breaks （DSBs） are introduced in cells by ionizing radiation and reactive oxygen species. In addition, they are commonly generated during V（D）J recombination, an essential aspect of the developing immune system. Failure to effectively repair these DSBs can result in chromosome breakage, cell death, onset of cancer, and defects in the immune system of higher vertebrates. Fortunately, all mammalian cells possess two enzymatic pathways that mediate the repair of DSBs： homologous recombination and non-homologous end-joining （NHEJ）. The NHEJ process utilizes enzymes that capture both ends of the broken DNA molecule, bring them together in a synaptic DNA-protein complex, and finally repair the DNA break. In this review, all the known enzymes that play a role in the NHEJ process are discussed and a working model for the co-operation of these enzymes during DSB repair is presented.     

Non-homologous DNA end joining

DNA double strand breaks (DSB) are the most serious form of DNA damage. Repair of DSBs is important to prevent chromosomal fragmentation, translocations and deletions. Non-homologous end joining (NHEJ) is one of three major pathways for the repair of DSBs in human cells. In this process two DNA ends are joined directly, usually with no sequence homology, although in the case of same polarity of the single stranded overhangs in DSBs, regions of microhomology are utilized. NHEJ is typically imprecise, a characteristic that is useful for immune diversification in lymphocytes in V(D)J recombination. The main components of the NHEJ system in eukaryotes are the catalytic subunit of DNA protein kinase (DNA-PKcs), Ku proteins, XRCC4, DNA ligase IV, and Artemis. This review focuses on the mechanisms an dregulation of DSB repair by NHEJ in mammalian cells.     

Repair of DNA double strand breaks by non-homologous end joining

DNA double strand breaks (DSB) are the most serious form of DNA damage. If not repaired they can lead to cell death. If misrepaired DSBs contribute to chromosomal aberrations and genomic instability. Non-homologous end joining (NHEJ) is one of two major pathways for the repair of DSBs in human cells. Proteins known to be required for NHEJ include the DNA-dependent protein kinase (DNA-PK), XRCC4, DNA ligase IV, and Artemis. This review discusses how these and other accessory proteins may function in the repair of DSBs produced by ionizing radiation (IR) and by V(D)J recombination.     

RAG proteins shepherd double-strand breaks to a specific pathway, suppressing error-prone repair, but RAG nicking initiates homologous recombination

The two major pathways for repairing double-strand breaks (DSBs), homologous recombination and nonhomologous end joining (NHEJ), have traditionally been thought to operate in different stages of the cell cycle. This division of labor is not absolute, however, and precisely what governs the choice of pathway to repair a given DSB has remained enigmatic. We pursued this question by studying the site-specific DSBs created during V(D)J recombination, which relies on classical NHEJ to repair the broken ends. We show that mutations that form unstable RAG postcleavage complexes allow DNA ends to participate in both homologous recombination and the error-prone alternative NHEJ pathway. By abrogating a key function of the complex, these mutations reveal it to be a molecular shepherd that guides DSBs to the proper pathway. We also find that RAG-mediated nicks efficiently stimulate homologous recombination and discuss the implications of these findings for oncogenic chromosomal rearrangements, evolution, and gene targeting.     

Pathways of DNA double-strand break repair during the mammalian cell cycle

Little is known about the quantitative contributions of nonhomologous end joining (NHEJ) and homologous recombination (HR) to DNA double-strand break (DSB) repair in different cell cycle phases after physiologically relevant doses of ionizing radiation. Using immunofluorescence detection of gamma-H2AX nuclear foci as a novel approach for monitoring the repair of DSBs, we show here that NHEJ-defective hamster cells (CHO mutant V3 cells) have strongly reduced repair in all cell cycle phases after 1 Gy of irradiation. In contrast, HR-defective CHO irs1SF cells have a minor repair defect in G(1), greater impairment in S, and a substantial defect in late S/G(2). Furthermore, the radiosensitivity of irs1SF cells is slight in G(1) but dramatically higher in late S/G(2), while V3 cells show high sensitivity throughout the cell cycle. These findings show that NHEJ is important in all cell cycle phases, while HR is particularly important in late S/G(2), where both pathways contribute to repair and radioresistance. In contrast to DSBs produced by ionizing radiation, DSBs produced by the replication inhibitor aphidicolin are repaired entirely by HR. irs1SF, but not V3, cells show hypersensitivity to aphidicolin treatment. These data provide the first evaluation of the cell cycle-specific contributions of NHEJ and HR to the repair of radiation-induced versus replication-associated DSBs.     

Alternative pathways for the repair of RAG-induced DNA breaks

RAG1 and RAG2 cleave DNA to generate blunt signal ends and hairpin coding ends at antigen receptor loci in lymphoid cells. During V(D)J recombination, repair of these RAG-generated double-strand breaks (DSBs) by the nonhomologous end-joining (NHEJ) pathway contributes substantially to the antigen receptor diversity necessary for immune system function, although recent evidence also supports the ability of RAG-generated breaks to undergo homology-directed repair (HDR). We have determined that RAG-generated chromosomal breaks can be repaired by pathways other than NHEJ in mouse embryonic stem (ES) cells, although repair by these pathways occurs at a significantly lower frequency than NHEJ. HDR frequency was estimated to be >or=40-fold lower than NHEJ frequency for both coding end and signal end reporters. Repair by single-strand annealing was estimated to occur at a comparable or lower frequency than HDR. As expected, V(D)J recombination was substantially impaired in cells deficient for the NHEJ components Ku70, XRCC4, and DNA-PKcs. Concomitant with decreased NHEJ, RAG-induced HDR was increased in each of the mutants, including cells lacking DNA-PKcs, which has been implicated in hairpin opening. HDR was increased to the largest extent in Ku70-/- cells, implicating the Ku70/80 DNA end-binding protein in regulating pathway choice. Thus, RAG-generated DSBs are typically repaired by the NHEJ pathway in ES cells, but in the absence of NHEJ components, a substantial fraction of breaks can be efficiently channeled into alternative pathways in these cells.     

Structural insights into NHEJ: Building up an integrated picture of the dynamic DSB repair super complex,one component and interaction at a time

Non-homologous end joining (NHEJ) is the major pathway for repair of DNA double-strand breaks (DSBs) in human cells. NHEJ is also needed for V(D)J recombination and the development of T and B cells in vertebrate immune systems, and acts in both the generation and prevention of non-homologous chromosomal translocations, a hallmark of genomic instability and many human cancers. X-ray crystal structures, cryo-electron microscopy envelopes, and small angle X-ray scattering (SAXS) solution conformations and assemblies are defining most of the core protein components for NHEJ: Ku70/Ku80 heterodimer; the DNA dependent protein kinase catalytic subunit (DNA-PKcs); the structure-specific endonuclease Artemis along with polynucleotide kinase/phosphatase (PNKP), aprataxin and PNKP related protein (APLF); the scaffolding proteins XRCC4 and XLF (XRCC4-like factor); DNA polymerases, and DNA ligase IV (Lig IV). The dynamic assembly of multi-protein NHEJ complexes at DSBs is regulated in part by protein phosphorylation. The basic steps of NHEJ have been biochemically defined to require: (1) DSB detection by the Ku heterodimer with subsequent DNA-PKcs tethering to form the DNA-PKcs-Ku-DNA complex (termed DNA-PK), (2) lesion processing, and (3) DNA end ligation by Lig IV, which functions in complex with XRCC4 and XLF. The current integration of structures by combined methods is resolving puzzles regarding the mechanisms, coordination and regulation of these three basic steps. Overall, structural results suggest the NHEJ system forms a flexing scaffold with the DNA-PKcs HEAT repeats acting as compressible macromolecular springs suitable to store and release conformational energy to apply forces to regulate NHEJ complexes and the DNA substrate for DNA end protection, processing, and ligation.     

Hairpin opening and overhang processing by an Artemis/DNA-dependent protein kinase complex in nonhomologous end joining and V(D)J recombination

Mutations in the Artemis protein in humans result in hypersensitivity to DNA double-strand break-inducing agents and absence of B and T lymphocytes (radiosensitive severe combined immune deficiency [RS-SCID]). Here, we report that Artemis forms a complex with the 469 kDa DNA-dependent protein kinase (DNA-PKcs) in the absence of DNA. The purified Artemis protein alone possesses single-strand-specific 5' to 3' exonuclease activity. Upon complex formation, DNA-PKcs phosphorylates Artemis, and Artemis acquires endonucleolytic activity on 5' and 3' overhangs, as well as hairpins. Finally, the Artemis:DNA-PKcs complex can open hairpins generated by the RAG complex. Thus, DNA-PKcs regulates Artemis by both phosphorylation and complex formation to permit enzymatic activities that are critical for the hairpin-opening step of V(D)J recombination and for the 5' and 3' overhang processing in nonhomologous DNA end joining.     

Dynamics of the Artemis and DNA-PKcs Complex in the Repair of Double-Strand Breaks

Pathologic chromosome breaks occur in human dividing cells ～10 times per day, and physiologic breaks occur in each lymphoid cell many additional times per day. Nonhomologous DNA end joining (NHEJ) is the major pathway for the repair of all of these double-strand breaks (DSBs) during most of the cell cycle. Nearly all broken DNA ends require trimming before they can be suitable for joining by ligation. Artemis is the major nuclease for this purpose. Artemis is tightly regulated by one of the largest protein kinases, which tethers Artemis to its surface. This kinase is called DNA-dependent protein kinase catalytic subunit (or DNA-PKcs) because it is only active when it encounters a broken DNA end. With this activation, DNA-PKcs permits the Artemis catalytic domain to enter a large cavity in the center of DNA-PKcs. Given this remarkably tight supervision of Artemis by DNA-PKcs, it is an appropriate time to ask what we know about the Artemis:DNA-PKcs complex, as we integrate recent structural information with the biochemistry of the complex and how this relates to other NHEJ proteins and to V(D)J recombination in the immune system.     

Evidence for Ku70/Ku80 association with full-length RAG1

Antigen receptor genes are assembled by a site-specific DNA rearrangement process called V(D)J recombination. This process proceeds through two distinct phases: a cleavage phase in which the RAG1 and RAG2 proteins introduce DNA double-strand breaks at antigen receptor gene segments, and a joining phase in which the resulting DNA breaks are processed and repaired via the non-homologous end-joining (NHEJ) repair pathway. Genetic and biochemical evidence suggest that the RAG proteins play an active role in guiding the repair of DNA breaks introduced during V(D)J recombination to the NHEJ pathway. However, evidence for specific association between the RAG proteins and any of the factors involved in NHEJ remains elusive. Here we present evidence that two components of the NHEJ pathway, Ku70 and Ku80, interact with full-length RAG1, providing a biochemical link between the two phases of V(D)J recombination.