Non-Homologous End-Joining, a Sticky Affair

DC van Gent1 and M van der Burg2

1Department of Cell Biology and Genetics, Erasmus MC, University Medical Center Rotterdam, Rotterdam, The Netherlands and 2Department of Immunology, Erasmus MC, University Medical Center Rotterdam, Rotterdam, The Netherlands

Rejoining of broken chromosomes is crucial for cell 2007). Homologous recombination uses the information survival and prevention of malignant transformation. Most on the sister chromatid or the homologous chromosome mammalian cells rely primarily on the non-homologous to align both DNA ends. In mammalian cells, this repair end-joining pathway of DNA double-strand break (DSB) pathway is probably restricted to the S and G2 phases of repair to accomplish this task. This review focuses both on the cell cycle when a sister chromatid is present. Non- the core non-homologous end-joining machinery, which homologous end-joining (NHEJ) does not require a consists of DNA-dependent protein kinase and the ligase homologous DNA template, although short patches of IV/XRCC4 complex, and on accessory factors that homologous nucleotides (microhomologies of one or a facilitate rejoining of a subset of the DSBs. We discuss few base pairs) are frequently used to align the DNA how the ATMprotein kinase and the Mre11/Rad50/Nbs1 ends. This review focuses on the mechanism of NHEJ complex might function in DSB repair and what role and the implications of defects in this repair pathway. ionizing radiation-induced foci may play in this process. Oncogene (2007) 26, 7731–7740; doi:10.1038/sj.onc.1210871

Keywords: DNA repair; V(D)J recombination; DNA Outline of the NHEJ pathway double-strand break repair; severe combined immunodeficiency; DNA damage response; chromosomal instability The NHEJ reaction starts with binding of the Ku70/80 heterodimer to a DNA end (Figure 1) (Weterings and van Gent, 2004). DNA end bound Ku70/80 then attracts the catalytic subunit of the DNA-dependent protein kinase (DNA-PKCS) and activates its protein kinase The DNA damage response activity (Smith and Jackson, 1999). Although many different targets have been identified, the major function DNA damage induces several responses, including DNA for this phosphorylation activity appears to be regulation repair, cell cycle checkpoints and apoptosis (Zhou and of the NHEJ reaction by DNA-PKCS autophosphoryla- Elledge, 2000; Bartek and Lukas, 2007). This is tion (Chan et al., 2002; Ding et al., 2003; Weterings et al., collectively referred to as the DNA damage response 2003). This reaction takes place after juxtaposition of (DDR). DNA double-strand breaks (DSBs) pose a DNA ends and is required for proper regulation of DNA particularly severe threat to genome stability, since lack end accessibility for other NHEJ proteins (Meek et al., of repair may cause loss of chromosome fragments 2007). Compatible DNA ends can now be joined directly during mitosis, whereas coupling of the wrong DNA by the ligase IV/XRCC4 complex. This reaction is ends is the basis for chromosomal translocations that stimulated by the recently discovered XLF/Cernunnos can induce neoplastic transformation (Burma et al., protein, which interacts with XRCC4 (Ahnesorg et al., 2006). To prevent loss of genetic material during 2006; Buck et al., 2006). However, in many circum- stances the DNA ends are not compatible. Ionizing mitosis, cells halt their cell cycle at the G2/M boundary until the majority of DSBs is repaired. On the other radiation, for example, creates a large number of ends hand, excessively damaged cells can be eliminated by that contain damaged bases and/or DNA backbone activation of apoptotic pathways, thereby preventing sugars that need processing before ligation. Therefore, survival of cells that might have lost proper growth NHEJ can accommodate nucleases, DNA polymerases regulation, for example by loss of heterozygosity of a polynucleotide kinase and other enzymes that render tumor suppressor gene. such ends ligatable by the ligase IV/XRCC4 complex. The most important factor for cell survival is repair of NHEJ is not only important for repair of DSBs that the DNA damage. DSBs can be repaired by two result from exogenous and endogenous DNA damaging fundamentally different mechanisms (Helleday et al., agent, but also for repair of DSBs generated during V(D)J recombination, which takes place during B- and T-cell differentiation to generate antigen specific recep- Correspondence: Dr DC van Gent, Department of Cell Biology and Genetics, Erasmus MC, PO Box 2040, Rotterdam 3000 CA, The tors (Jung et al., 2006). Variable (V), Diversity (D) and Netherlands. Joining (J) segments in Immunoglobulin (Ig) and T Cell E-mail: [email protected] Receptor (TCR) genes are joined to form the mature Non-homologous end-joining DC van Gent and M van der Burg 7732 is called the SAP domain (Aravind and Koonin, 2000). This domain has been found to bind to DNA in some other proteins and may also have a function as an accessory DNA binding domain in the Ku heterodimer, although it is not in close proximity to DNA in the crystal structure. However, this domain is connected to the rest of the Ku70 polypeptide with a flexible linker arm, which could facilitate considerable movement. The Ku80 C-terminal domain has been found to influence interaction with DNA-PKCS (Gell and Jackson, 1999; Singleton et al., 1999; Falck et al., 2005). Interaction between Ku70/80 and DNA-PKCS requires a DNA end. The assembled DNA-PK complex then acquires the ability to phosphorylate a number of target proteins, but the functional relevance of most targets is not clear. However, DNA-PKCS autophosphorylation is clearly important for the NHEJ reaction (Chan et al., 2002; Ding et al., 2003; Weterings et al., 2003). It is accomplished in trans, after formation of a synaptic complex that brings together the two sides of the DSB (Meek et al., 2007). Several clusters of autophosphorylation sites have been mapped. A cluster of seven sites between residues 2609 and 2647 has a major influence on remodeling the DNA-PK complex to make the DNA ends accessible for ligation (Ding et al., 2003; Block et al., 2004; Reddy et al., 2004). Conversely, phosphorylation in the region between residues 2023– 2056 appears to be involved in reducing access of nuclease activities to the DNA ends (Chen et al., 2005; Cui et al., 2005), while phosphorylation of residue 3950 Figure 1 Non-homologous end-joining model. After double-strand also influences the joining efficiency (Douglas et al., break formation, the Ku70/80 heterodimer binds to the DNA ends 2007). These autophosphorylation sites have been and attracts DNA-PKCS. This activates the DNA-PK kinase activity, which leads to autophosphorylation, which enables the subsequent mapped after in vitro phosphorylation reactions and it processing and ligation steps. The small triangle symbolizes a DNA is not completely clear whether phosphorylation at these end that needs processing before ligation. sites requires DNA-PK activity in vivo; Ataxia-Telan- giectasia mutated protein kinase (ATM) may also V(D)J exon in B and T cells, respectively (Schatz, 2004). participate in a subset of these events (Chen et al., 2005). The RAG1 and RAG2 proteins create DSBs at the However, the importance of autophosphorylation has border between conserved Recombination Signal Se- clearly been shown by mutagenesis of the phosphorylation quences (RSSs) and the coding DNA segments, creating sites, as well as the kinase domain in the DNA-PKCS gene blunt DNA ends at the RSS and coding ends with a (Kurimasa et al., 1999). hairpin structure in which the top strand is covalently Juxtaposition of the correct DNA ends is an obligatory coupled to the bottom strand (McBlane et al., 1995; van step in the NHEJ process. The Ku70/80 heterodimer is Gent et al., 1996). These DSBs require the NHEJ necessary for this process, but DNA-PKCS is also machinery for their repair. required to form a stable synaptic complex in which both ends are held together. Such end-to-end junctions have been observed by Scanning Force Microscopy and cryo electron microscopy (Yaneva et al., 1997; Spagnolo DNA-PK et al., 2006). Biochemical experiments have shown that DNA-PKCS is indeed required for stable juxtaposition of The structure of the Ku70/80 heterodimer directly shows DNA ends (DeFazio et al., 2002; Drouet et al., 2005). why this protein binds specifically to DNA ends: its ring However, DNA-PKCS is not absolutely required for all shape allows it to thread onto a double-stranded DNA end-joining events, because blunt DNA ends are joined molecule, but not to internal DNA positions (Walker with relatively high efficiency in DNA-PKCS deficient et al., 2001). As expected on the basis of this structure, cells (Lieber et al., 1988; van Heemst et al., 2004) and the protein can slide along a DNA molecule to internal DSB repair after ionizing radiation is much more efficient positions without the need for an energy source, such as in these cells than in Ku80 or XRCC4 deficient cells ATP (de Vries et al., 1989; Ristic et al., 2003). The ring (Riballo et al., 2004). shape is made of approximately the N-terminal 500 Although Ku70/80 binds DNA ends quite tightly, amino-acid residues of Ku70 and Ku80. The Ku70 C Ku70/80 as well as DNA-PKCS exchange from DSBs terminus shows homology to a number of proteins and within a few minutes in living cells (Mari et al., 2006;

Oncogene Non-homologous end-joining DC van Gent and M van der Burg 7733 Uematsu et al., 2007). The dynamics of NHEJ complexes complementary DNA ends, but does not seem to be an are influenced dramatically by DNA-PK autophosphor- absolute requirement for the majority of DSBs (Gu et al., ylation: a DNA-PKCS mutant protein without kinase 2007; Tsai et al., 2007; Wu et al., 2007). activity or with mutations in the autophosphorylation sites exchanged much more slowly than the wild type protein. The dynamic nature of these complexes is somewhat unexpected: as repair of approximately 75% Processing factors for NHEJ of the DSBs takes 1 h, most NHEJ complexes fall apart The core NHEJ factors described above are sufficient to before repair has been completed. It is presently not clear join compatible DNA ends that have a 50 phosphate and how this affects the chance to create chromosomal 0 aberrations, but it is to be expected that dissociation of a3 hydroxyl. However, most DSB inducing agents, such as ionizing radiation, create a large variety of DNA NHEJ complexes will be a prerequisite for formation of ends that need processing before they can be ligated deletions and translocations. (Nikjoo et al., 1998). For this purpose, mammals have a large toolbox of enzymes to cope with all these damages. Damaged DNA termini often require removal of one or a few damaged bases from the end before ligation can DNA ligase IV, XRCC4 and XLF/Cernunnos proceed. The Artemis nuclease was discovered as the gene that is mutated in the majority of radiosensitive The final step in the NHEJ pathway is ligation of DNA TÀBÀ severe combined immunodeficiency (SCID) patients ends by the DNA ligase IV/XRCC4 complex. NHEJ (Moshous et al., 2001). Its function in V(D)J recombina- efficiency is minimal in the absence of this complex and a tion is opening of the coding end DNA hairpin structures defect in ligase IV or XRCC4 is even lethal in mice that are formed by RAG1/2 cleavage at Recombination (Barnes et al., 1998; Frank et al., 1998; Gao et al., 1998b). et al et al XRCC4 is absolutely required for ligase IV activity, Signal Sequences (Ma ., 2002; Pannicke ., 2004; Poinsignon et al., 2004). Artemis needs DNA-PK for its probably because the ligase IV protein needs it for activity and DNA-PK phosphorylates Artemis, suggesting stability and correct targeting to DSBs (Grawunder et al., 1997; Bryans et al., 1999; Chen et al., 2000; Teo and that Artemis phosphorylation by DNA-PK might be important (Ma et al., 2005). However, mutation of all Jackson, 2000; Hsu et al., 2002; Drouet et al., 2005; phosphorylation sites in the Artemis gene did not result in Costantini et al., 2007). The three dimensional structure diminished activity, whereas DNA-PK autophosphoryla- of the largest part of the XRCC4 protein has been tion was shown to be required for Artemis activation determined (Junop et al., 2000). The most remarkable (Goodarzi et al., 2006). Although the types of lesions that feature is a long a-helical stalk that gives the protein an need Artemis mediated processing have not yet been extended structure, which would be ideal to serve as a flexible linker arm that could connect the ligase to the rest identified, it is clear that a subset of 15–20% of ionizing radiation-induced DSBs remain unrepaired for several of the NHEJ complex. Ligase IV interacts with the days in Artemis deficient cells (Riballo et al., 2004; Wang extended a-helix of XRCC4 via a domain in the C et al et al terminus that contains two BRCT domains (Grawunder ., 2005; Darroudi ., 2007). Another feature of many DSBs is non-complementarity et al., 1998; Sibanda et al., 2001). The XRCC4 protein because of damaged or missing bases. Therefore, DNA exists under most conditions as a dimer and can form ends often require synthesis of a limited number of tetramers by interaction of two dimers via the region in nucleotides as part of the NHEJ process. Although short the a-helix that also binds ligase IV (Modesti et al., 2003). single-stranded gaps may be filled in by many different Although the function of XRCC4 tetramerization is not DNA polymerases, addition of nucleotides to a DSB is clear, this interaction might strengthen interactions probably restricted to the Y-type DNA polymerases polm between two NHEJ complexes on juxtaposed DNA ends. and poll, which have partially overlapping specificities Interaction between Ku70/80 and the DNA ligase IV/ (Lee et al., 2004; Nick McElhinny et al., 2005; Capp et al., XRCC4 complex is important for efficient NHEJ (Nick McElhinny et al., 2000). Interactions of both the ligase 2006, 2007). To permit both DNA polymerization and ligation, a 30-OH and a 50-phosphate need to be present at component and the XRCC4 polypeptide with the Ku the DNA ends. Polynucleotide kinase is recruited to DNA heterodimer have been described, which may all be necessary for efficient DNA end ligation (Chen et al., 2000; Hsu et al., endsbyinteractionwithXRCC4andprovidestheactivity to both remove 30-phosphates and add 50-phosphates 2002; Drouet et al., 2005; Mari et al., 2006; Costantini et al., (Koch et al., 2004). 2007). DNA-PKCS provides even additional stability to NHEJ complexes through interactions with XRCC4, although the ligase complex does not absolutely require Other accessory factors for NHEJ the catalytic subunit for accumulation at DSBs. XLF/Cernunnos is the most recently identified NHEJ Several other factors have been found to be required for factor. The gene was cloned independently from immuno- repair of the same subset of DSBs that require Artemis deficient patients that showed radio sensitivity (Buck et al., (Riballo et al., 2004). These include the MRE11/ 2006) and by a yeast 2-hybrid screen for proteins that RAD50/NBS1 (MRN) complex, MDC1, 53BP1 and interact with XRCC4 (Ahnesorg et al., 2006). It stimulates the ATM protein kinase, which were previously found NHEJ, which appears to be mainly important for non- to be involved in DNA damage-induced cell cycle

Oncogene Non-homologous end-joining DC van Gent and M van der Burg 7734 checkpoints and apoptosis (Lavin and Kozlov, 2007). An important clue for the MRN function in DSB repair may come from the peculiar structure of this protein complex. The RAD50 component of this complex has two very long, flexible coiled-coil arms that contain a Zinc hook at the tip (Hopfner et al., 2002). In the presence of Zn2 þ , this structure forms a bridge between two RAD50 arms, suggesting that the MRN complex may be involved in keeping two DNA ends in close proximity. Juxtaposition of DNA ends through MRN interactions has indeed been observed using purified proteins and Scanning Force Microscopy (de Jager et al., 2001; Moreno-Herrero et al., 2005) and this protein complex has been found to accumulate in large nuclear foci within minutes after DSB formation (Maser et al., 1997). Although the function of these foci is not clear, several mutants that do support focus formation show DSB repair defects. They have been hypothesized to support accumulation of a high local concentration of repair proteins. However, in light of the specific structure of the MRN complex, one could also imagine, that MRN (probably together with other proteins) forms a microenvironment that holds together the two DNA ends. Such a ‘sticky ball’ would allow some degree of freedom for movement of DNA ends and access of (NHEJ) proteins within this structure (Figure 2a). MRN foci formation requires several other factors, including the MDC1 and 53BP1 proteins and phosphorylation of histone variant H2AX (Celeste et al., 2003; Ward et al., 2003; Xu and Stern, 2003; Lee et al., 2005). As the ATM protein kinase is the major H2AX kinase after induction of DSBs, the main function of ATM could be creation of a chromatin environment that facilitates MRN accumulation in nuclear foci. This notion is supported by the observation, that cells deficient in all these repair factors have similar repair defects, most notably increased levels of residual DSBs after ionizing radiation and chromosomal instability (Ward et al., 2003; Franco et al., 2006; Lou et al., 2006; Lavin and Kozlov, 2007). However, it is not directly clear why these foci forming factors would be required for the same subset of DSBs that require processing by Artemis. They might all be involved in activating the Artemis activity, but all Figure 2 Possible functions of accessory factors. (a) A double- biochemical evidence shows that Artemis nuclease strand break induces ATM kinase activity, which phosphorylates histone H2AX. Subsequently, MDC1, 53BP1 and the MRN depends only on DNA-PK activity. Therefore, we complex bind to the phosphorylated nucleosomes. Finally, MRN would like to propose a different explanation for this complexes interact with each other via their Zinc hooks at the phenomenon (Figure 2b). As described above, Ku70/80 RAD50 coiled coil arms that protrude from the central protein head. Gray ellipses depict nucleosomes that contain H2AX, white and DNA-PKCS are in dynamic equilibrium between the DNA bound and free states. This implies that DNA ellipses are nucleosomes with other H2A variants. (b) After DSB formation, the non-homologous end-joining (NHEJ) proteins and ends loose their NHEJ complexes every few minutes. In MRN accumulate more or less independently on the DNA. When principle, free DNA ends might loose juxtaposition only NHEJ proteins hold the ends together, the DNA ends can unless some other structure keeps them together. We either be joined directly or the NHEJ proteins can dissociate from propose, that the ‘sticky ball’ of MRN complexes may the complex, which can lead to loss of juxtaposition of the ends. However, when the ‘sticky ball’ of MRN is present, the ends will be carry out this function. Obviously, the chance to loose held in close proximity, even when the NHEJ complex falls apart. the other end of the DSB in the absence of the ‘sticky Although each protein molecule in the ‘sticky ball’ can exchange ball’ is higher for DNA ends that require more time for rapidly with protein molecules in solution, the structure stays joining, for example, because of processing. Therefore, intact. It is not yet clear which step in this process should happen the subset of DSBs that requires Artemis would largely first, but we expect that the order of events is not fixed. coincide with DSBs that need the ‘sticky ball’ to keep them juxtaposed.

Oncogene Non-homologous end-joining DC van Gent and M van der Burg 7735 Alternative end-joining pathways recombination deficiency results from a defect in NHEJ. The majority of these patients have mutations in Artemis Although the major end-joining pathway relies on DNA- (Moshous et al., 2001; Li et al., 2002; Kobayashi et al., PK and ligase IV, backup pathways can support a low 2003; Noordzij et al., 2003). The clinical phenotype of level of end joining. This backup system relies mainly on RAG1, RAG2 and Artemis-deficient patients is similar patches of a few base pairs of (micro)homology, that may and can be mimicked in mice (Table 1). A partial V(D)J assist in alignment of the ends (Kabotyanski et al., 1998; recombination defect caused by hypomorphic mutations in Verkaik et al., 2002). Although microhomology-mediated RAG1, RAG2 or Artemis leads to the Omenn’s syndrome, end-joining is not very efficient for V(D)J recombination which is characterized by prominent generalized papular and repair of blunt DSBs (van Heemst et al., 2004), this skin eruption, eosinophilia, hyper IgE, circulating oligo- backup pathway can mediate Ig heavy chain class switch clonal T-cells in addition to the failure to thrive and recombination in B cells rather efficiently (Yan et al., opportunistic infections (Villa et al., 1998; Ege et al., 2005). 2007). However, this microhomology driven backup Other cases of hypomorphic mutations in Artemis have system appears to be quite error-prone, as chromosomal been associated with partial T- and B-cell immunodeficiency translocations have frequently been found to contain with the development of aggressive EBV-associated B-cell microhomologies at the junctions. The existence of lymphomas (Moshous et al., 2003). backup end-joining pathways may explain why chromo- Mutations in the DNA ligase IV gene are also somal translocations have been found regularly in cells associated with immunodeficiencies. The clinical spec- with defects in NHEJ components, although this process trum of these patients is quite diverse, varying from obviously requires joining of DNA ends (Iliakis et al., radiosensitive leukemia, via the LIG4 syndrome (that is 2004; Burma et al., 2006). characterized by immunodeficiency, microcephaly and growth retardation) to radiosensitive SCID (Riballo et al., 1999; O’Driscoll et al., 2001; van der Burg et al., 2006). As DNA ligase IV knockout mice are embryonic Intersection of NHEJ with other cellular processes lethal, the LIG4 mutations found in patients are most probably hypomorphic. The different types of mutations The DNA damage response includes not only repair, but might explain the variation in clinical phenotype, also regulation of cell-cycle progression and apoptosis. although differences in other genes could also contribute These processes are intimately connected and several to this variability. Finally, mutations in the ligase- proteins have functions at multiple levels. For example, associated XLF/Cernunnos gene have been found in the Ku70 protein is not only involved in NHEJ as part patients with growth retardation, microcephaly and of the Ku70/80 heterodimer, but it also influences immunodeficiency due to profound T and B-cell apoptosis by interaction with the proapoptotic Bax lymphocytopenia (Buck et al., 2006). Other NHEJ protein (Sawada et al., 2003). On the other hand, the components have not been found mutated in human cell-cycle checkpoint protein kinase ATM is most SCID patients yet, but based on the phenotype of the probably also directly involved in repair of a subset of XRCC4, Ku70, Ku80 and DNA-PK knockout mice, DSBs (Riballo et al., 2004), showing that many proteins CS mutations in these genes are also expected to result function in more than one genetic pathway. clinically in SCID. Furthermore, most NHEJ proteins have been found Although most defects described above result in to interact with telomeres. Obviously, these chromo- TÀBÀSCID, more careful characterization of these some ends are DNA ends that should not be joined to patients revealed interesting differences. First, the another DNA end. However, the Ku70/80 heterodimer, NHEJ defect is characterized by studying DSB repair DNA-PK and the MRN complex have been found to CS kinetics, using gH2AX foci counting in primary preferentially localize to these structures (Bailey et al., fibroblasts after irradiation. For example, DNA ligase 1999; Slijepcevic, 2006). These interactions do not promote IV-deficient fibroblasts display slow DSB repair (van der end-joining and even seem to prevent end-to-end fusion of Burg et al., 2006), whereas Artemis-deficient cells show chromosomes. normal fast initial DSB repair, with 15–20% of DSB that are not repaired at all (Riballo et al., 2004; Wang et al., 2005; Darroudi et al., 2007). The precise analysis NHEJ and developmental defects in mice and men of V(D)J recombination products is also very informa- tive. V(D)J recombination substrates can be transfected NHEJ defects in mammals result in absence of B and T into patient cells (primary fibroblasts) or residual cells and give rise to SCID. Both cellular and humoral junctions from bone marrow precursor B-cells can be immunity are affected and these patients are clinically amplified (Table 2) (Verkaik et al., 2002; van der Burg characterized by opportunistic infections, protracted et al., 2006, 2007). Artemis deficiency causes normal diarrhea and failure to thrive, which becomes apparent signal joint formation, but a strong reduction in the during the first months of life (See also de Villartay et al. in numbers of coding joints, which are characterized by this issue). Mutations in the RAG1 and RAG2 genes long stretches of P-nucleotides due to aberrant hairpin account for B70% of all TÀBÀ SCID patients (Schwarz opening (Rooney et al., 2003; van der Burg et al., 2007). et al., 1996). The majority of the remaining patients show The junction characteristics are therefore indicative for hypersensitivity to ionizing radiation, because the V(D)J the affected V(D)J recombination component and the

Oncogene Non-homologous end-joining DC van Gent and M van der Burg 7736 Table 1 Consequences of NHEJ gene defects in men and mice Gene Human clinical phenotype Knock-out mouse phenotype Ref

DNA- — Radiosensitive SCID T-cell tumors (Gao et al. (1998a); Taccioli et al. PKCS (1998) Ku70 — Radiosensitive SCID Growth retardation Gu et al. (1997); Ouyang et al. (1997) T-cell tumors Ku80 — Radiosensitive SCID Growth retardation Nussenzweig et al. (1996); Zhu et al. (1996) XRCC4 — Embryonic lethal, apoptosis of Gao et al. (1998b) post-mitotic neurons LIG4 Radiosensitive leukemia or LIG4 Embryonic lethal, apoptosis of Barnes et al. (1998); Frank et al. syndrome characterized by growth post-mitotic neurons (1998); O’Driscoll et al. (2001); Riballo retardation, microcephaly and et al. (1999); van der Burg et al. (2006) immunodeﬁciency or radiosensitive SCID

Artemis Radiosensitive SCID Radiosensitive SCID (Moshous et al. (2001); Rooney et al. (2002)

XLF/Cer- Growth retardation, microcephaly, and ES cells: radiosensitive, impaired V(D)J (Buck et al. (2006); Zha et al. (2007) nunnos immunodeﬁciency due to profound T and recombination B-cell lymphocytopenia

Table 2 Characteristics of residual coding joints derived from characterized by loss of Purkinje cells, which results in precursor B-cells of healthy donors and SCID patients a progressive ataxia (Digweed et al., 1999; Chun and Affected step in V(D)J Mutated gene Na Pa Deletionsa Gatti, 2004; McKinnon, 2004). It is caused by mutations recombination in the ATM protein kinase, which functions not only in NHEJ, but also in maintaining cell-cycle checkpoints Normal V(D)J — 7.9 0.2 10.2 recombination and apoptotic responses. Nijmegen Breakage syndrome Initiation RAG1/RAG2 7.7 0.3 12.1 (NBS) shares several cellular characteristics with AT, Hairpin opening Artemis 4.0 6.7 3.3 although these patients do not show ataxia. Instead they Ligation LIG4 2.8 0.2 28.2 display microcephaly and a mild mental retardation. Again, it is not clear whether the NHEJ defect adds to Abbreviations: N, non-templated; P, palindromic van der Burg et al. (2006, the brain phenotype of these patients. Finally, AT like 2007). aAverage numbers of nucleotides per coding joint (DH-JH). disorder results from hypomorphic mutations in MRE11 and is very similar to AT (Taylor et al., 2004).

integration of all results facilitates recognition of new genetic defects. Finally, identification of novel NHEJ factors has been NHEJ and carcinogenesis shown by de Villartay and co-workers (Moshous et al., 2001; Buck et al., 2006). They developed approaches to Most tumors show chromosomal instability, which may clone genes involved in NHEJ by complementation of add to their malignant phenotype. However, most mouse radiosensitive fibroblasts using a cDNA expression models with deficiencies in NHEJ genes are not extremely library approach and selection schemes that make use cancer prone, although chromosomal instability at the of the NHEJ defect. The combination of these chromosomal level is severely increased. Apparently, approaches has been very successful for identification other cellular safeguards (‘gate keepers’) prevent malig- of mutations in patients as well as gaining knowledge nant transformation. Indeed, combination of the NHEJ about the mechanism of NHEJ. defect with p53 deficiency leads to extremely fast DNA repair defects can also have impact on neuronal development of B-cell tumors in mice (van Gent et al., development. NHEJ is critical for differentiating cells to 2001). Even reduced NHEJ capacity can facilitate prevent apoptosis of the post-mitotic neurons (Lee and malignant transformation in a checkpoint-deficient ge- McKinnon, 2007). This is particularly clear in DNA netic background such as DNA ligase IV heterozygosity ligase IV and XRCC4-deficient mice (Frank et al., 1998; in ink4a/arfÀ/À mice (Sharpless et al., 2001). Gao et al., 1998b), but Ku70 or Ku80 deficiency shows The dependence on various mechanisms to control similar defects to a lesser extent (Nussenzweig et al., tumor development is not equal in all cell types. For 1996; Gu et al., 1997). The neuropathy in patients with example, the ligase IV deficiency in a p53 deficient the LIG4 syndrome and XLF/Cernunnos deficiency is background results in lymphoid tumors (Frank et al., confined to characteristic facial features and microce- 2000), while ligase IV heterozygosity in the ink4a/arf phaly, suggesting that these mutations do not cause a deficient mice increases the incidence of soft-tissue complete NHEJ deficiency. sarcomas (Sharpless et al., 2001). The effect on genome Mutations in NHEJ accessory factors are more stability varies also between different cell types, for difficult to interpret. Ataxia telangiectasia (AT) is example heterozygosity in the Ku80, ligase IV or XRCC4

Oncogene Non-homologous end-joining DC van Gent and M van der Burg 7737 genes increased genomic instability in fibroblasts, but not In conclusion, decreased NHEJ capacity increases in cultured developing lymphocytes (Karanjawala et al., chromosomal instability, which is a hallmark of many 1999). tumors. However, very high levels of tumorigenesis In contrast to NHEJ defects, mutations of the ATM require also defects in cell-cycle checkpoints or apopto- and NBS1 genes increase tumorigenesis, even in the sis, which would otherwise prevent proliferation of cells absence of additional mutations. The repair defect in with DNA damage. A more profound knowledge of the ATM or NBS1 deficient cells resembles Artemis deficiency interactions between these ‘care taker’ and ‘gate keeper’ very closely, but tumorigenesis is much more pronounced functions will help in understanding the carcinogenic in AT or NBS patients. This can most probably be process and applying this knowledge to develop anti- explained by a combination of defects: both genes are not cancer treatments. only involved in NHEJ, but also in cell-cycle checkpoints and DSB-induced apoptosis (Deckbar et al., 2007; Lavin Acknowledgements and Kozlov, 2007). Furthermore, aberrant rejoining appears to be much more frequent in AT or NBS cells We thank Drs PO Mari, SP Perseng and NGJ Jaspers for than in Artemis deficient cells (Darroudi et al., 2007), useful comments. Research in our laboratories is supported by which can be explained by a defect in maintaining the European Union (Projects ‘RISC-RAD’ and ‘DNA juxtaposition of DNA ends, rather than an inability to Repair’) and the Dutch Organization for Scientific Research process the lesion in preparation for joining. (NWO/ZonMw veni grant 916.56.107).

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