Search

DNA damage response mechanisms encompass pathways of DNA repair, cell cycle checkpoint arrest and apoptosis. Together, these mechanisms function to maintain genomic stability in the face of exogenous and endogenous DNA damage. ATM is activated in response to double strand breaks and initiates cell cycle checkpoint arrest. Recent studies in human fibroblasts have shown that ATM also regulates a mechanism of end-processing that is required for a component of double strand break repair. Human fibroblasts rarely undergo apoptosis after ionising radiation and, therefore, apoptosis is not considered in our review. The dual function of ATM raises the question as to how the two processes, DNA repair and checkpoint arrest, interplay to maintain genomic stability. In this review, we consider the impact of ATM's repair and checkpoint functions to the maintenance of genomic stability following irradiation in G2. We discuss evidence that ATM's repair function plays little role in the maintenance of genomic stability following exposure to ionising radiation. ATM's checkpoint function has a bigger impact on genomic stability but strikingly the two damage response pathways co-operate in a more than additive manner. In contrast, ATM's repair function is important for survival post irradiation.

The Artemis nuclease is defective in radiosensitive severe combined immunodeficiency patients and is required for the repair of a subset of ionising radiation induced DNA double-strand breaks (DSBs) in an ATM and DNA-PK dependent process. Here, we show that Artemis phosphorylation by ATM and DNA-PK in vitro is primarily attributable to S503, S516 and S645 and demonstrate ATM dependent phosphorylation at serine 645 in vivo. However, analysis of multisite phosphorylation mutants of Artemis demonstrates that Artemis phosphorylation is dispensable for endonuclease activity in vitro and for DSB repair and V(D)J recombination in vivo. Importantly, DNA-dependent protein kinase catalytic subunit (DNA-PKcs) autophosphorylation at the T2609-T2647 cluster, in the presence of Ku and target DNA, is required for Artemis-mediated endonuclease activity. Moreover, autophosphorylated DNA-PKcs stably associates with Ku-bound DNA with large single-stranded overhangs until overhang cleavage by Artemis. We propose that autophosphorylation triggers conformational changes in DNA-PK that enhance Artemis cleavage at single-strand to double-strand DNA junctions. These findings demonstrate that DNA-PK autophosphorylation regulates Artemis access to DNA ends, providing insight into the mechanism of Artemis mediated DNA end processing.

Ataxia-telangiectasia mutated and Rad3 related (ATR)-Seckel syndrome and autosomal recessive primary microcephaly (MCPH) syndrome share clinical features. RNA interference (RNAi) of MCPH1 have implicated the protein it encodes as a DNA-damage response protein that regulates the transcription of Chk1 and BRCA1, two genes involved in the response to DNA damage. Here, we report that truncating mutations observed in MCPH-syndrome patients do not impact on Chk1 or BRCA1 expression or early ATR-dependent damage-induced phosphorylation events. However, like ATR-Seckel syndrome cells, MCPH1-mutant cell lines show defective G2-M checkpoint arrest and nuclear fragmentation after DNA damage, and contain supernumerary mitotic centrosomes. MCPH1-mutant and ATR-Seckel cells also show impaired degradation of Cdc25A and fail to inhibit Cdc45 loading onto chromatin after replication arrest. Additionally, microcephalin interacts with Chk1. We conclude that MCPH1 has a function downstream of Chk1 in the ATR-signalling pathway. In contrast with ATR-Seckel syndrome cells, MCPH1-mutant cells have low levels of Tyr 15-phosphorylated Cdk1 (pY15-Cdk1) in S and G2 phases, which correlates with an elevated frequency of G2-like cells displaying premature chromosome condensation (PCC). Thus, MCPH1 also has an ATR-independent role in maintaining inhibitory Cdk1 phosphorylation, which prevents premature entry into mitosis.

Null mutations in Artemis confer a condition described as RS-SCID, in which patients display radiosensitivity combined with severe combined immunodeficiency. Here, we characterize the defect in Artemis in a patient who displayed progressive combined immunodeficiency (CID) and elevated lymphocyte apoptosis. The patient is a compound heterozygote with novel mutations in both alleles, resulting in Artemis proteins with either L70 deletion or G126D substitution. Both mutational changes impact upon Artemis function and a fibroblast cell line derived from the patient (F96-224) has greatly reduced Artemis protein. In contrast to Artemis null cell lines, which fail to repair a subset of DNA double strand breaks (DSBs) induced by ionizing radiation, F96-224 cells show slow but residual DSB rejoining. Despite showing intermediate cellular and clinical features, F96-224 cells are as radiosensitive as Artemis null cell lines. We developed a FACS-based assay to examine cell division and cellular characteristics for 10 days following exposure to ionizing radiation (2 and 4 Gy). This analysis demonstrated that F96-224 cells show delayed cell death when compared with rapid growth arrest of an Artemis null cell line, and the emergence of a cycling population shown by a control line. F96-224 cells also display elevated chromosome aberrations when compared with control cells. F96-224 therefore represents a novel phenotype for a hypomorphic cell line. We suggest that delayed cell death contributes to the progressive CID phenotype of the Artemis patient.

The efficient repair of DNA double-strand breaks is crucial in safeguarding the genomic integrity of organisms. Responses to double-strand breaks include complex signal-transduction, cell-cycle-checkpoint and repair pathways. Defects in these pathways lead to several human disorders with pleiotropic clinical features. Dissection of the molecular basis that underlies the diverse clinical features is enhancing our understanding of the damage-response mechanisms and their role in development, and might ultimately facilitate treatment.

ATM is a central component of a signal transduction process that responds to DNA double strand breaks (DSBs) ultimately effecting cell cycle checkpoint arrest and/or apoptosis. Recent studies have shown that ATM also regulates a mechanism of processing a subset of DNA ends that appear to be difficult to ligate, since they are rejoined with slow kinetics in control cells. In the absence of this process, which involves the nuclease, Artemis, the DSBs either remain unrejoined or potentially undergo misrejoining. Thus, ATM's checkpoint function specifically facilitates its repair function. Here, we discuss the contribution of this novel function of ATM to survival after ionising irradiation and to cancer avoidance. We suggest that ATM's strength as a damage response protein lies in the co-ordination of its repair and checkpoint functions making a razor sharp knife out of two blunter edges.

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.

We examined telomere maintenance in cells of 11 primary fibroblast cell lines with differing genetic defects that confer sensitivity to ionizing radiation. These included cell lines derived from patients with ataxia telangiectasia, Nijmegen breakage syndrome, Fanconi anemia, defective Artemis, DNA ligase I and DNA ligase IV, an immunodeficient patient with a defect in DNA double-strand break repair, and a patient diagnosed with xeroderma pigmentosum who, in addition, showed severe clinical sensitivity to ionizing radiation. Our results, based on Southern blot, flow-FISH and Q-FISH (quantitative FISH) measurements, revealed an accelerated rate of telomere shortening in most cell lines derived from the above patients compared to cell lines from normal individuals or a cell line isolated from a heterozygotic parent of one radiosensitive patient. This accelerated telomere shortening was accompanied by the formation of chromatin bridges in anaphase cells, indicative of the early loss of telomere capping function and in some cases low levels of chromosome abnormalities in metaphase cells. We also analyzed telomere maintenance in mouse embryonic stem cells deficient in Brca1, another defect that confers radiosensitivity. Similarly, these cells showed accelerated telomere shortening and mild telomere dysfunction in comparison to control cells. Our results suggest that mechanisms that confer sensitivity to ionizing radiation may be linked with mechanisms that cause telomere dysfunction.

Ataxia telangiectasia mutated protein (ATM) is a damage response kinase that initiates a signal transduction response to the presence of DNA double strand breaks (DSBs) regulating cell cycle checkpoint arrest and apoptosis. Indirect evidence has argued that A-T cells also harbour a repair defect since unrepaired DSBs can be observed in non-replicating A-T cells after ionising radiation (IR). The basis underlying such a repair defect has remained unexplained, however. Artemis, a nuclease, whose activity is modified by phosphorylation in vitro, was recently identified as a novel ATM substrate. Artemis and ATM function in a common pathway required for the processing of a subset of double stranded DNA ends induced by IR prior to rejoining by non-homologous end-joining (NHEJ). This subset of DSBs are those normally rejoined with slow kinetics. Additional components of the ATM signal transduction pathway, Nbs1, Mre11, H2AX and 53BP1, are also required for this component of DSB repair. This process substantially contributes to survival post irradiation. Our findings add a new dimension to the ATM signal transduction response demonstrating ATM-dependent regulation of an end-processing mechanism that functions during the cell cycle delay effected by ATM.

DNA nonhomologous end-joining in vivo requires the DNA-dependent protein kinase (DNA-PK) and DNA ligase IV/XRCC4 (LX) complexes. Here, we have examined the impact of histone octamers and linker histone H1 on DNA end-joining in vitro. Packing of the DNA substrate into dinucleosomes does not significantly inhibit ligation by LX. However, LX ligation activity is substantially reduced by the incorporation of linker histones. This inhibition is independent of the presence of core histone octamers and cannot be restored by addition of Ku alone but can be partially rescued by DNA-PK. The kinase activity of DNA-PK is essential for the recovery of end-joining. DNA-PK efficiently phosphorylates histone H1. Phosphorylated histone H1 has a reduced affinity for DNA and a decreased capacity to inhibit end-joining. Our findings raise the possibility that DNA-PK may act as a linker histone kinase by phosphorylating linker histones in the vicinity of a DNA break and coupling localized histone H1 release from DNA ends, with the recruitment of LX to carry out double-stranded ligation. Thus, by using histone H1-bound DNA as a template, we have reconstituted the end-joining step of DNA nonhomologous end-joining in vitro with a requirement for DNA-PK.

Double-strand breaks (DSBs) arise endogenously during normal cellular processes and exogenously by genotoxic agents such as ionizing radiation (IR). DSBs are one of the most severe types of DNA damage, which if left unrepaired are lethal to the cell. Several different DNA repair pathways combat DSBs, with nonhomologous end-joining (NHEJ) being one of the most important in mammalian cells. Competent NHEJ catalyses repair of DSBs by joining together and ligating two free DNA ends of little homology (microhomology) or DNA ends of no homology. The core components of mammalian NHEJ are the catalytic subunit of DNA protein kinase (DNA-PK(cs)), Ku subunits Ku70 and Ku80, Artemis, XRCC4 and DNA ligase IV. DNA-PK is a nuclear serine/threonine protein kinase that comprises a catalytic subunit (DNA-PK(cs)), with the Ku subunits acting as the regulatory element. It has been proposed that DNA-PK is a molecular sensor for DNA damage that enhances the signal via phosphorylation of many downstream targets. The crucial role of DNA-PK in the repair of DSBs is highlighted by the hypersensitivity of DNA-PK(-/-) mice to IR and the high levels of unrepaired DSBs after genotoxic insult. Recently, DNA-PK has emerged as a suitable genetic target for molecular therapeutics such as siRNA, antisense and novel inhibitory small molecules. This review encompasses the recent literature regarding the role of DNA-PK in the protection of genomic stability and focuses on how this knowledge has aided the development of specific DNA-PK inhibitors, via both small molecule and directed molecular targeting techniques. This review promotes the inhibition of DNA-PK as a valid approach to enhance the tumor-cell-killing effects of treatments such as IR.

Nijmegen breakage syndrome (NBS) is characterised by microcephaly, developmental delay, characteristic facial features, immunodeficiency and radiosensitivity. Nbs1, the protein defective in NBS, functions in ataxia telangiectasia mutated protein (ATM)-dependent signalling likely facilitating ATM phosphorylation events. While NBS shares overlapping characteristics with ataxia telangiectasia, it also has features overlapping with ATR-Seckel (ATR: ataxia-telangiectasia and Rad3-related protein) syndrome, a subclass of Seckel syndrome mutated in ATR. We show that Nbs1 also facilitates ATR-dependent phosphorylation. NBS cell lines show a similar defect in ATR phosphorylation of Chk1, c-jun and p-53 in response to UV irradiation- and hydroxyurea (HU)-induced replication stalling. They are also impaired in ubiquitination of FANCD2 after HU treatment, which is ATR dependent. Following HU-induced replication arrest, NBS and ATR-Seckel cells show similarly impaired G2/M checkpoint arrest and an impaired ability to restart DNA synthesis at stalled replication forks. Moreover, NBS cells fail to retain ATR in the nucleus following HU treatment and extraction. Our findings suggest that Nbs1 functions in both ATR- and ATM-dependent signalling. We propose that the NBS clinical features represent the result of these combined defects.

To date, the only reported genetic defect identified in the developmental disorder, Seckel syndrome, is a mutation in ataxia telangiectasia and Rad3-related protein (ATR). Seckel syndrome is clinically and genetically heterogeneous and whether defects in ATR significantly contribute to Seckel syndrome is unclear. Firstly, we characterize ATR-Seckel cells for their response to DNA damage. ATR-Seckel cells display impaired phosphorylation of ATR-dependent substrates, impaired G2/M checkpoint arrest and elevated micronucleus (MN) formation following exposure to UV and agents that cause replication stalling. We describe a novel phenotype, designated nuclear fragmentation (NF), that occurs following replication arrest. Finally, we report that ATR-Seckel cells have an endogenously increased number of centrosomes in mitotic cells demonstrating a novel role for ATR in regulating centrosome stability. We exploit these phenotypes to examine cell lines derived from additional unrelated Seckel syndrome patients. We show that impaired phosphorylation of ATR-dependent substrates is a common but not invariant feature of Seckel syndrome cell lines. In contrast, all cell lines displayed defective G2/M arrest, increased levels of NF and MN formation following exposure to agents that cause replication stalling. All the Seckel syndrome cell lines examined showed increased endogenous centrosome numbers. Though ATR cDNA can complement the defects in ATR-Seckel cells, it failed to complement any of the additional cell lines. We conclude that Seckel syndrome represents a further damage response disorder that is uniquely associated with defects in the ATR-signalling pathway resulting in failed checkpoint arrest following exposure to replication fork stalling.

The hereditary disorder ataxia telangiectasia (A-T) is associated with striking cellular radiosensitivity that cannot be attributed to the characterized cell cycle checkpoint defects. By epistasis analysis, we show that ataxia telangiectasia mutated protein (ATM) and Artemis, the protein defective in patients with RS-SCID, function in a common double-strand break (DSB) repair pathway that also requires H2AX, 53BP1, Nbs1, Mre11, and DNA-PK. We show that radiation-induced Artemis hyperphosphorylation is ATM dependent. The DSB repair process requires Artemis nuclease activity and rejoins approximately 10% of radiation-induced DSBs. Our findings are consistent with a model in which ATM is required for Artemis-dependent processing of double-stranded ends with damaged termini. We demonstrate that Artemis is a downstream component of the ATM signaling pathway required uniquely for the DSB repair function but dispensable for ATM-dependent cell cycle checkpoint arrest. The significant radiosensitivity of Artemis-deficient cells demonstrates the importance of this component of DSB repair to survival.

Fanconi anemia (FA), an autosomal recessive chromosomal instability syndrome, is characterized clinically by developmental abnormalities, growth retardation, progressive bone marrow failure, pancytopenia, and pronounced cancer predisposition. Nijmegen Breakage Syndrome (NBS) is a related disorder that shares overlapping clinical features, principally, developmental delay, microcephaly, and cancer predisposition. The diagnosis has relied on chromosomal instability following exposure to DNA cross-linking agents in FA and to ionizing radiation (IR) in NBS. We describe two patients who clinically had FA, but showed sensitivity to both DNA cross-linking agents and ionizing radiation, and who were found to have a rare mutation in the NBS gene. The importance of genetic diagnosis with respect to treatment and prognosis is discussed.

LIG4 syndrome patients have hypomorphic mutations in DNA ligase IV. Although four of the five identified patients display immunodeficiency and developmental delay, one patient was developmentally normal. The developmentally normal patient had the same homozygous mutation (R278H) in DNA ligase IV as one of the more severely affected patients, who additionally had two linked polymorphisms. Here, we examine the impact of the mutations and polymorphisms identified in the LIG4 syndrome patients. Examination of recombinant mutant proteins shows that the severity of the clinical features correlates with the level of residual ligase activity. The polymorphisms decrease the activity of DNA ligase IV by approximately 2-fold. When combined with the otherwise mild R278H mutation, the activity is reduced to a level similar to other LIG4 patients who display immunodeficiency and developmental delay. This demonstrates how coupling of a mutation and polymorphism can have a marked impact on protein function and provides an example where a polymorphism may have influenced clinical outcome. Analysis of additional mutational changes in LIG4 syndrome (R580X, R814X and G469E) have led to the identification of a nuclear localization signal in DNA ligase IV and sites impacting upon DNA ligase IV adenylation.

Around 15-20 hereditary disorders associated with impaired DNA damage response mechanisms have been previously described. The range of clinical features associated with these disorders attests to the significant role that these pathways play during development. Recently, three new such disorders have been reported extending the importance of the damage response pathways to human health. LIG4 syndrome is conferred by hypomorphic mutations in DNA ligase IV, an essential component of DNA non-homologous end-joining (NHEJ), and is associated with pancytopaenia, developmental and growth delay and dysmorphic facial features. Radiosensitive severe combined immunodeficiency (RS-SCID) is caused by mutations in Artemis, a protein that plays a subsidiary role in non-homologous end-joining although it is not an essential component. RS-SCID is characterised by severe combined immunodeficiency but patients have no overt developmental abnormalities. ATR-Seckel syndrome is caused by mutations in ataxia telangiectasia and Rad3 related protein (ATR), a component of a DNA damage signalling pathway. ATR-Seckel syndrome patients have dramatic microcephaly and marked growth and developmental delay. The clinical features of these patients are considered in the light of the function of the defective protein.

H2AX phosphorylation is an early step in the response to DNA damage. It is widely accepted that ATM (ataxia telangiectasia mutated protein) phosphorylates H2AX in response to DNA double-strand breaks (DSBs). Whether DNA-dependent protein kinase (DNA-PK) plays any role in this response is unclear. Here, we show that H2AX phosphorylation after exposure to ionizing radiation (IR) occurs to similar extents in human fibroblasts and in mouse embryo fibroblasts lacking either DNA-PK or ATM but is ablated in ATM-deficient cells treated with LY294002, a drug that specifically inhibits DNA-PK. Additionally, we show that inactivation of both DNA-PK and ATM is required to ablate IR-induced H2AX phosphorylation in chicken cells. We confirm that H2AX phosphorylation induced by DSBs in nonreplicating cells is ATR (ataxia telangiectasia and Rad3-related protein) independent. Taken together, we conclude that under most normal growth conditions, IR-induced H2AX phosphorylation can be carried out by ATM and DNA-PK in a redundant, overlapping manner. In contrast, DNA-PK cannot phosphorylate other proteins involved in the checkpoint response, including chromatin-associated Rad17. However, by phosphorylating H2AX, DNA-PK can contribute to the presence of the damage response proteins MDC1 and 53BP1 at the site of the DSB.

The Sleeping Beauty (SB) element is a useful tool to probe transposon-host interactions in vertebrates. We investigated requirements of DNA repair factors for SB transposition in mammalian cells. Factors of nonhomologous end joining (NHEJ), including Ku, DNA-PKcs, and Xrcc4 as well as Xrcc3/Rad51C, a complex that functions during homologous recombination, are required for efficient transposition. NHEJ plays a dominant role in repair of transposon excision sites in somatic cells. Artemis is dispensable for transposition, consistent with the lack of a hairpin structure at excision sites. Ku physically interacts with the SB transposase. DNA-PKcs is a limiting factor for transposition and, in addition to repair, has a function in transposition that is independent from its kinase activity. ATM is involved in excision site repair and affects transposition rates. The overlapping but distinct roles of repair factors in transposition and in V(D)J recombination might influence the outcomes of these mechanistically similar processes.

The ATM protein, which is mutated in individuals with ataxia telangiectasia (AT), is central to cell cycle checkpoint responses initiated by DNA double-strand breaks (DSBs). ATM's role in DSB repair is currently unclear as is the basis underlying the radiosensitivity of AT cells. We applied immunofluorescence detection of gamma-H2AX nuclear foci and pulsed-field gel electrophoresis to quantify the repair of DSBs after X-ray doses between 0.02 and 80 Gy in confluence-arrested primary human fibroblasts from normal individuals and patients with mutations in ATM and DNA ligase IV, a core component of the nonhomologous end-joining (NHEJ) repair pathway. Cells with hypomorphic mutations in DNA ligase IV exhibit a substantial repair defect up to 24 h after treatment but continue to repair for several days and finally reach a level of unrepaired DSBs similar to that of wild-type cells. Additionally, the repair defect in NHEJ mutants is dose dependent. ATM-deficient cells, in contrast, repair the majority of DSBs with normal kinetics but fail to repair a subset of breaks, irrespective of the initial number of lesions induced. Significantly, after biologically relevant radiation doses and/or long repair times, the repair defect in AT cells is more pronounced than that of NHEJ mutants and correlates with radiosensitivity. NHEJ-defective cells analyzed for survival following delayed plating after irradiation show substantial recovery while AT cells fail to show any recovery. These data argue that the DSB repair defect underlies a significant component of the radiosensitivity of AT cells.

Upon DNA damage, p53-binding protein 1 (53BP1) relocalizes to sites of DNA double-strand breaks and forms discrete nuclear foci, suggesting its role in DNA damage responses. We show that 53BP1 changed its localization from the detergent soluble to insoluble fraction after treatment of cells with x-ray, but not with ultraviolet or hydroxyurea. Either DNase or phosphatase treatment of the insoluble fraction released 53BP1 into the soluble fraction, showing that 53BP1 binds to chromatin in a phosphorylation-dependent manner after X-irradiation of cells. 53BP1 was retained at discrete nuclear foci in X-irradiated cells even after detergent extraction of cells, showing that the chromatin binding of 53BP1 occurs at sites of DNA double-strand breaks. The minimal domain for focus formation was identified by immunofluorescence staining of cells ectopically expressed with 53BP1 deletion mutants. This domain consisted of conserved Tudor and Myb motifs. The Tudor plus Myb domain possessed chromatin binding activity in vivo and bound directly to both double-stranded and single-stranded DNA in vitro. This domain also stimulated end-joining by DNA ligase IV/Xrcc4, but not by T4 DNA ligase in vitro. We conclude that 53BP1 has the potential to participate directly in the repair of DNA double-strand breaks.

The DNA ligase IV.XRCC4 complex (LX) functions in DNA non-homologous-end joining, the main pathway for double-strand break repair in mammalian cells. We show that, in contrast to ligation by T4 ligase, the efficiency of LX ligation of double-stranded (ds) ends is critically dependent upon the length of the DNA substrate. The effect is specific for ds ligation, and LX/DNA binding is not influenced by the substrate length. Ku stimulates LX ligation at concentrations resulting in 1-2 Ku molecules bound per substrate, whereas multiply Ku-bound DNA molecules inhibit ds ligation. The combined footprint of DNA with Ku and LX bound is the sum of each individual footprint suggesting that the two complexes are located in tandem at the DNA end. Inhibition of Ku translocation by the presence of cis-platinum adducts on the DNA substrate severely inhibits ligation by LX. Fluorescence resonance energy transfer analysis using fluorophore-labeled Ku and DNA molecules showed that, as expected, Ku makes close contact with the DNA end and that addition of LX can disrupt this close contact. Finally, we show that recruitment of LX by Ku is impaired in an adenylation-defective mutant providing further evidence that LX interacts directly with the DNA end, possibly via the 5'-phosphate as shown for prokaryotic ligases. Taken together, our results suggest that, when LX binds to a Ku-bound DNA molecule, it causes inward translocation of Ku and that freedom to move inward on the DNA is essential to Ku stimulation of LX activity.

Seckel syndrome (OMIM 210600) is an autosomal recessive disorder characterized by intrauterine growth retardation, dwarfism, microcephaly and mental retardation. Clinically, Seckel syndrome shares features in common with disorders involving impaired DNA-damage responses, such as Nijmegen breakage syndrome (OMIM 251260) and LIG4 syndrome (OMIM 606593). We previously mapped a locus associated with Seckel syndrome to chromosome 3q22.1-q24 in two consanguineous Pakistani families. Further marker analysis in the families, including a recently born unaffected child with a recombination in the critical region, narrowed the region to an interval of 5 Mbp between markers D3S1316 and D3S1557 (145.29 Mbp and 150.37 Mbp). The gene encoding ataxia-telangiectasia and Rad3-related protein (ATR) maps to this region. A fibroblast cell line derived from an affected individual displays a defective DNA damage response caused by impaired ATR function. We identified a synonymous mutation in affected individuals that alters ATR splicing. The mutation confers a phenotype including marked microcephaly (head circumference 12 s.d. below the mean) and dwarfism (5 s.d. below the mean). Our analysis shows that UV-induced ATR activation can occur in non-replicating cells following processing by nucleotide excision repair.

DNA nonhomologous end-joining (NHEJ) is the major pathway for repairing DNA double-strand breaks in mammalian cells. It also functions to carry out rearrangements at the specialized breaks introduced during V(D)J recombination. Here, we describe a patient with T(-)B(-) severe combined immunodeficiency, whose cells have defects closely resembling those of NHEJ-defective rodent cells. Cells derived from this patient show dramatic radiosensitivity, decreased double-strand break rejoining, and reduced fidelity in signal and coding joint formation during V(D)J recombination. Detailed examination indicates that the patient is defective neither in the known factors involved in NHEJ in mammals (Ku70, Ku80, DNA-dependent protein kinase catalytic subunit, Xrcc4, DNA ligase IV, or Artemis) nor in the Mre11/Rad50/Nbs1 complex, whose homologue in Saccharomyces cerevisiae functions in NHEJ. These results provide strong evidence that additional activities are crucial for NHEJ and V(D)J recombination in mammals.

Chromosomal instability plays a pivotal role in multistep carcinogenesis by facilitating the acquisition of the multiple genetic alterations necessary for malignant transformation. In order to study the role of abnormal DNA repair in malignant melanoma, we measured the ability of cell lines from malignant melanoma and that of primary melanocytes to process 4 different kinds of DNA damage (pyrimidine dimers, oxidative DNA lesions, replication errors, and DNA double-strand breaks) using 4 different plasmid assays. Based on the number of chromosomes, the DNA index, and the rates of spontaneous micronuclei, the chromosomal stability in primary melanocytes and the melanoma line LIBR was characterized as being high, intermediate in M1, and low in MeWo. Repair of UVB photoproducts, of oxidative DNA damage, and of replication errors was not impaired in any melanoma line. Using linearized shuttle vector plasmid pZ189, LIBR cells and primary melanocytes exhibited a high efficiency of joining overlapping DNA ends, reflecting proficient repair of DNA double-strand breaks. Joining efficiency was reduced slightly in M1 and 2.9-fold in MeWo. This indicates that in the melanoma cell lines studied here, an increase in chromosomal instability is accompanied by a pronounced impairment in the ability to join DNA ends. Although a small sample was studied, this raises the possibility that defects in DNA end joining may also contribute to genetic instability and chromosome aberrations in melanoma., (Copyright 2003 S. Karger AG, Basel)

The DNA double stranded break (DSB) repair mechanism, non-homologous end joining (NHEJ) represents an essential step in antigen receptor gene rearrangement mechanisms, processes believed to be intimately involved in the aetiology of lymphoproliferative disease. We investigated the potential impact that previously undescribed polymorphisms identified within NHEJ DNA ligase IV (LIG4) have upon predisposition to several lymphoproliferative disorders, including leukaemia, lymphoma, and multiple myeloma. Two LIG4 polymorphisms were examined, both C>T transitions, which result in the amino acid substitutions A3V and T9I. Inheritance of the LIG4 A3V CT genotype was found to be significantly associated with a two-fold reduction in risk of developing multiple myeloma (OR 0.49, 95% CI 0.27 to 0.89). Similarly, inheritance of the LIG4 T9I CT and the T9I TT genotypes were found to associate with a 1.5-fold reduction (OR 0.77, 95% CI 0.51 to 1.17) and a four-fold reduction (OR 0.22, 95% CI 0.07 to 0.70) in risk of developing multiple myeloma respectively, suggesting a gene dosage effect for this polymorphism. The LIG4 A3V and T9I variant alleles are in linkage disequilibrium (D'=0.95, p<0.0001), and the protective effect associated with these polymorphisms was found to be the result of inheritance of the A3V-T9I CT and A3V-T9I TT haplotypes. These data suggest that genetic variants of NHEJ LIG4 may modulate predisposition to multiple myeloma, a tumour characterised by aberrant immunoglobulin (Ig) class switch recombination.

In eukaryotic cells, double-strand breaks (DSBs) in DNA are generally repaired by the pathway of homologous recombination or by DNA nonhomologous end joining (NHEJ). Both pathways have been highly conserved throughout eukaryotic evolution, but no equivalent NHEJ system has been identified in prokaryotes. The NHEJ pathway requires a DNA end-binding component called Ku. We have identified bacterial Ku homologs and show that these proteins retain the biochemical characteristics of the eukaryotic Ku heterodimer. Furthermore, we show that bacterial Ku specifically recruits DNA ligase to DNA ends and stimulates DNA ligation. Loss of these proteins leads to hypersensitivity to ionizing radiation in Bacillus subtilis. These data provide evidence that many bacteria possess a DNA DSB repair apparatus that shares many features with the NHEJ system of eukarya and suggest that this DNA repair pathway arose before the prokaryotic and eukaryotic lineages diverged.

In response to ionizing radiation (IR), the tumor suppressor p53 is stabilized and promotes either cell cycle arrest or apoptosis. Chk2 activated by IR contributes to this stabilization, possibly by direct phosphorylation. Like p53, Chk2 is mutated in patients with Li-Fraumeni syndrome. Since the ataxia telangiectasia mutated (ATM) gene is required for IR-induced activation of Chk2, it has been assumed that ATM and Chk2 act in a linear pathway leading to p53 activation. To clarify the role of Chk2 in tumorigenesis, we generated gene-targeted Chk2-deficient mice. Unlike ATM(-/-) and p53(-/-) mice, Chk2(-/-) mice do not spontaneously develop tumors, although Chk2 does suppress 7,12-dimethylbenzanthracene-induced skin tumors. Tissues from Chk2(-/-) mice, including those from the thymus, central nervous system, fibroblasts, epidermis, and hair follicles, show significant defects in IR-induced apoptosis or impaired G(1)/S arrest. Quantitative comparison of the G(1)/S checkpoint, apoptosis, and expression of p53 proteins in Chk2(-/-) versus ATM(-/-) thymocytes suggested that Chk2 can regulate p53-dependent apoptosis in an ATM-independent manner. IR-induced apoptosis was restored in Chk2(-/-) thymocytes by reintroduction of the wild-type Chk2 gene but not by a Chk2 gene in which the sites phosphorylated by ATM and ataxia telangiectasia and rad3(+) related (ATR) were mutated to alanine. ATR may thus selectively contribute to p53-mediated apoptosis. These data indicate that distinct pathways regulate the activation of p53 leading to cell cycle arrest or apoptosis.

Cell lines from Nijmegen Breakage Syndrome (NBS) and ataxia telangiectasia (A-T) patients show defective S phase checkpoint arrest. In contrast, only A-T but not NBS cells are significantly defective in radiation-induced G1/S arrest. Phosphorylation of some ATM substrates has been shown to occur in NBS cells. It has, therefore, been concluded that Nbs1 checkpoint function is S phase specific. Here, we have compared NBS with A-T cell lines (AT-5762ins137) that express a low level of normal ATM protein to evaluate the impact of residual Nbs1 function in NBS cells. The radiation-induced cell cycle response of these NBS and 'leaky' A-T cells is almost identical; normal G2/M arrest after 2 Gy, intermediate G1/S arrest depending on the dose and an A-T-like S phase checkpoint defect. Thus, the checkpoint assays differ in their sensitivity to low ATM activity. Radiation-induced phosphorylation of the ATM-dependent substrates Chk2, RPAp34 and p53-Ser15 are similarly impaired in AT-5762ins137 and NBS cells in a dose dependent manner. In contrast, NBS cells show normal ability to activate ATM kinase following irradiation in vitro and in vivo. We propose that Nbs1 facilitates ATM-dependent phosphorylation of multiple downstream substrates, including those required for G1/S arrest.

Ionising radiation (IR) induces a range of DNA damage similar to that which arises endogenously from reactive oxygen species generated as by-products of metabolism. However, due to non-homogeneous energy deposition, the damage from IR frequently occurs in clusters producing unique 'complex' lesions. Cells have evolved a range of mechanisms to respond to DNA damage, which include pathways of DNA repair and processes that prevent the proliferation of damaged cells. However, the repair mechanisms are not fool proof and clustered radiation-induced lesions pose a particular problem. Whether DNA damage created by IR can be repaired accurately, mis-repaired or not repaired at all is of utmost importance in considering the impact of radiation exposure. Here, the current knowledge is discussed of the repair of double strand breaks, a biologically important lesion induced by IR, in the context of the fidelity of the repair mechanisms and the consequences of mis-repair or lack of repair.

DNA damage response mechanisms serve to protect cells from exogenous and endogenous DNA damaging agents with the aim of maintaining genomic stability. In contrast, the generation of an efficient immune response requires the creation of a repertoire of distinct immunoglobulin and T cell receptor genes able to recognise the huge array of antigens that may be encountered in a lifetime. Surprisingly, cells have exploited the same mechanisms used to maintain genomic integrity to create genetic diversity during immune development. Here, we review the damage response mechanisms operating on DNA double strand breaks and their function during development of the immune response. We discuss disorders that are associated with immunodeficiency and defective responses to the presence of DNA double strand breaks.

DNA ligase IV functions in DNA nonhomologous end-joining and V(D)J recombination. Four patients with features including immunodeficiency and developmental and growth delay were found to have mutations in the gene encoding DNA ligase IV (LIG4). Their clinical phenotype closely resembles the DNA damage response disorder, Nijmegen breakage syndrome (NBS). Some of the mutations identified in the patients directly disrupt the ligase domain while others impair the interaction between DNA ligase IV and Xrcc-4. Cell lines from the patients show pronounced radiosensitivity. Unlike NBS cell lines, they show normal cell cycle checkpoint responses but impaired DNA double-strand break rejoining. An unexpected V(D)J recombination phenotype is observed involving a small decrease in rejoining frequency coupled with elevated imprecision at signal junctions.

DNA ligase IV functions in DNA non-homologous end-joining, in V(D)J recombination, and during brain development. We previously reported a homozygous mutation (R278H) in DNA ligase IV in a developmentally normal leukemia patient who overresponded to radiotherapy. The impact of this hypomorphic mutation has been evaluated using cellular, biochemical, and structural approaches. Structural modeling using T7 DNA ligase predicts that the activity and conformational stability of the protein is likely to be impaired. We show that wild type DNA ligase IV-Xrcc4 is an efficient double-stranded ligase with distinct optimal requirements for adenylate complex formation versus rejoining. The mutation impairs the formation of an adenylate complex as well as reducing the rejoining activity. Additionally, it imparts temperature-sensitive activity to the protein consistent with the predictions of the structural modeling. At the cellular level, the mutation confers a unique V(D)J recombination phenotype affecting the fidelity of signal joint formation with little effect on the frequency of the reaction. These findings suggest that hypomorphic mutations in ligase IV may allow normal development but confer marked radiosensitivity.

The DNA-dependent protein kinase (DNA-PK), comprised of the Ku70/Ku80 (now known as G22p1/Xrcc5) heterodimer and the catalytic subunit DNA-PKcs (now known as Prkdc), is required for the nonhomologous end joining (NHEJ) pathway of DNA double-strand break repair. The mechanism of action of DNA-PK remains unclear. We have investigated whether DNA-PK regulates gene transcription in vivo after DNA damage using the subtractive hybridization technique of cDNA representational difference analysis (cDNA RDA). Differential transcription, both radiation-dependent and independent, was detected and confirmed in primary mouse embryo fibroblasts from DNA-PKcs(-/-) and DNA-PKcs(+/+) mice. We present evidence that transcription of the extracellular matrix gene laminin alpha 4 (Lama4) is regulated by DNA-PK in a radiation-independent manner. However, screening of both primary and immortalized DNA-PKcs-deficient cell lines demonstrates that the majority of differences were not consistently dependent on DNA-PK status. Similar results were obtained in experiments using KU mutant hamster cell lines, indicating heterogeneity of transcription between closely related cell lines. Our results suggest that while DNA-PK may be involved in limited gene-specific transcription, it does not play a major role in the transcriptional response to DNA damage.

Background: Fanconi anaemia is a rare disease associated with cellular sensitivity to chemicals (e.g. mitomycin C and diepoxybutane); variable but mild cellular radiosensitivity has also been reported., Materials and Methods: A 32-year-old patient with Fanconi anaemia and tonsillar carcinoma, treated by radiotherapy, was found to exhibit profound clinical radiosensitivity. Confluent, ulcerating oropharyngeal mucositis developed after a conventionally fractionated dose of 34Gy and healing was incomplete by 2 months after cessation of therapy., Results: Cellular radiosensitivity assays and RPLD studies from this patient did not suggest any major detectable radiosensitivity., Conclusion: There is a discrepancy between the observed clinical radiosensitivity and the usual "predictive" radiosensitivity assays in this patient with Fanconi anaemia.

Two major pathways for repairing DNA double-strand breaks (DSBs) have been identified in mammalian cells, nonhomologous end-joining (NHEJ) and homologous recombination (HR). Inactivation of NHEJ is known to lead to an elevated level of spontaneous and radiation-induced chromosomal rearrangements associated with an increased risk of tumorigenesis. This has raised the idea of a caretaker role for NHEJ. It is, however, not known whether NHEJ itself can also cause rearrangements. To investigate, on the DNA level, the influence of a defect in NHEJ on the formation of genomic rearrangements, we applied an assay based on Southern hybridization that allows the identification and quantification of incorrectly rejoined DSB ends produced by ionizing radiation. After 80 Gy of X-irradiation at a high dose rate (23 Gy/min), wild-type cells repaired 50% of the induced DSBs within 24 h by incorrect rejoining. This frequency of DSB misrejoining is considerably reduced in NHEJ-deficient cells. Low-dose-rate experiments, in which the cells were exposed to 80 Gy over a period of 14 days under repair conditions, led to no detectable misrejoining in wild-type cells but revealed a misrejoining frequency of 10% in NHEJ-deficient cells. This shows that in situations of separated breaks, NHEJ deficiency leads to genomic rearrangements, in agreement with chromosomal studies. However, if multiple DSBs coincide, even wild-type cells form genomic rearrangements frequently. These repair events are absent in Ku80-, DNA-PKcs-, and DNA ligase IV-deficient cells but are present in RAD54(-/-) cells. This strongly suggests that NHEJ has, in addition to its caretaker role, also the potential to effect genomic rearrangements. We propose that it serves as an efficient pathway for rejoining correct break ends in situations of separated breaks but generates genomic rearrangements if DSBs are close in time and space.

DNA double strand break (DSB) repair by non-homologous end joining (NHEJ) in mammalian cells requires the Ku70-Ku80 heterodimer, the DNA-PK catalytic subunit DNA-PKcs, as well as DNA ligase IV and Xrcc4. NHEJ of plasmid DSBs in Saccharomyces cerevisiae requires Ku, Xrcc4 and DNA ligase IV, as well as Mre11, Rad50, Xrs2 and DNA damage checkpoint proteins. Saccharomyces cerevisiae Ku is also required for telomere length maintenance and transcriptional silencing. We have characterized NHEJ in Schizosaccharomyces pombe using an extrachromosomal assay and find that, as anticipated, it is Ku70 and DNA ligase IV dependent. Unexpectedly, we find that Rad32, Rad50 (the S.pombe homologues of Mre11 and Rad50, respectively) and checkpoint proteins are not required for NHEJ. Furthermore, although S.pombe Ku70 is required for maintenance of telomere length, it is dispensable for transcriptional silencing at telomeres and is located throughout the nucleus rather than concentrated at the telomeres. Together, these results provide insight into the mechanism of NHEJ and contrast significantly with recent studies in S.cerevisiae.

Cells derived from Nijmegen Breakage Syndrome (NBS) patients display radiosensitivity and cell cycle checkpoint defects. Here, we examine whether the radiosensitivity of NBS cells is the result of a repair defect or whether it can be attributed to impaired checkpoint arrest. We report a small increased fraction of unrejoined double strand breaks and, more significantly, increased chromosome breaks in noncycling NBS cells at 24 h after irradiation. One of the NBS lines examined (347BR) was atypical in showing a nearly normal checkpoint response. In contrast to the mild checkpoint defect, 347BR displays marked y-ray sensitivity similar to that shown by other NBS lines. Thus, the gamma-ray sensitivity correlates with the repair defect rather than impaired checkpoint control. Taken together, the results provide direct evidence for a repair defect in NBS cells and are inconsistent with the suggestion that the radiosensitivity is attributable only to impaired checkpoint arrest. 347BR also displays elevated spontaneous damage that cannot be attributed to impaired G2-M arrest, suggesting a function of Nbsl in decreasing or limiting the impact of spontaneously arising double strand breaks.

DNA-PKcs, the catalytic subunit of DNA-dependent protein kinase (DNA-PK), has a phosphoinositol 3-kinase (PI 3-K) domain close to its C-terminus. Cell lines derived from the SCID mouse have been utilised as a model DNA-PKcs-defective system. The SCID mutation results in truncation of DNA-Pkcs at the extreme C-terminus leaving the PI 3-K domain intact. The mutated protein is expressed at low levels in most SCID cell lines, leaving open the question of whether the mutation abolishes kinase activity. Here, we show that a SCID cell line that expresses the mutant protein normally has dramatically impaired kinase activity. We estimate that the residual kinase activity typically present in SCID fibroblast cell lines is at least two orders of magnitude less than that found in control cells. Our results substantiate evidence that DNA-PKcs kinase activity is required for DSB rejoining and V(D)J recombination and show that the extreme C-terminal region of DNA-PKcs, present in PI 3-K-related protein kinases but absent in bona fide PI 3 lipid kinases, is required for DNA-PKcs to function as a protein kinase. We also show that expression of mutant DNA-PKcs protein confers a growth disadvantage, providing an explanation for the lack of DNA-PKcs expression in most SCID cell lines.

Thermal radiosensitization is thought to result from inhibition of repair of radiation-induced DNA damage, DNA double-strand breaks in particular. Since the DNA-dependent protein kinase (DNA-PK) complex plays a major role in the nonhomologous end-joining of DSBs, it has been suggested that inactivation of this complex as a whole or of its individual subunits by heat might be involved in radiosensitization by heat. To test this hypothesis further, the ability of heat to enhance the radiosensitivity of cells proficient or deficient in either Ku80 or the DNA-PK catalytic subunit (DNA-PKcs) was investigated. In cells of two Ku80-deficient and two DNA-PKcs-deficient and double-strand break-deficient cell lines, the extent of radiosensitization by heat was not reduced compared to that in both their isogenic gene-complemented counterparts as well as to that in their parental cells. Thus radiosensitization by hyperthermia can be obtained irrespective of the Ku80 or DNA-PKcs status in cells. Therefore, Ku80 or DNA-PKcs and hence nonhomologous DSB end-joining do not play a crucial role in the enhancement of cellular radiosensitivity by hyperthermia.

Severe immunodeficiency characterized by lymphopenia was found in two siblings, one of whom was examined in detail. The calcium flux, pattern of tyrosine phosphorylation of proteins, and interleukin 2 (IL-2) production and proliferation in response to mitogens suggested that the peripheral blood T cells activated normally. The peripheral blood T cells were shown to have an activated phenotype with increased expression of CD45RO+ and CD95/Fas. Increased spontaneous apoptosis occurred in unstimulated lymphocyte cultures. The elevated apoptosis was not due to alterations in expression or to mutations in Bcl-2, Bcl-X(L), or Flip, nor could the spontaneous apoptosis be prevented by blocking Fas, suggesting that it was independent of Fas signaling. This is the first inherited combined immunodeficiency associated with impaired lymphocyte survival. Fibroblasts derived from the patient showed appreciable radiosensitivity in clonal assays, but apoptosis was not elevated. Our results show that the fibroblasts represent a new radiosensitive phenotype not associated with cell cycle checkpoint defects, V(D)J recombination defects, or elevated chromosome breakage. We suggest that the affected gene plays a role in an undetermined damage response mechanism that results in elevated spontaneous apoptosis in lymphoid cells and radiosensitivity in fibroblasts.

The major mechanism for the repair of DNA double-strand breaks (DSBs) in mammalian cells is non-homologous end-joining (NHEJ), a process that involves the DNA-dependent protein kinase [1] [2], XRCC4 and DNA ligase IV [3] [4] [5] [6]. Rodent cells and mice defective in these components are radiation-sensitive and defective in V(D)J-recombination, showing that NHEJ also functions to rejoin DSBs introduced during lymphocyte development [7] [8]. 180BR is a radiosensitive cell line defective in DSB repair, which was derived from a leukaemia patient who was highly sensitive to radiotherapy [9] [10] [11]. We have identified a mutation within a highly conserved motif encompassing the active site in DNA ligase IV from 180BR cells. The mutated protein is severely compromised in its ability to form a stable enzyme-adenylate complex, although residual activity can be detected at high ATP concentrations. Our results characterize the first patient with a defect in an NHEJ component and suggest that a significant defect in NHEJ that leads to pronounced radiosensitivity is compatible with normal human viability and does not cause any major immune dysfunction. The defect, however, may confer a predisposition to leukaemia.

Damage to DNA in the cell activates the tumour-suppressor protein p53, and failure of this activation leads to genetic instability and a predisposition to cancer. It is therefore crucial to understand the signal transduction mechanisms that connect DNA damage with p53 activation. The enzyme known as DNA-dependent protein kinase (DNA-PK) has been proposed to be an essential activator of p53, but the evidence for its involvement in this pathway is controversial. We now show that the p53 response is fully functional in primary mouse embryonic fibroblasts lacking DNA-PK: irradiation-induced DNA damage in these defective fibroblasts induces a normal response of p53 accumulation, phosphorylation of a p53 serine residue at position 15, nuclear localization and binding to DNA of p53. The upregulation of p53-target genes and cell-cycle arrest also occur normally. The DNA-PK-deficient cell line SCGR11 contains a homozygous mutation in the DNA-binding domain of p53, which may explain the defective response by p53 reported in this line. Our results indicate that DNA-PK activity is not required for cells to mount a p53-dependent response to DNA damage.

Ku is a heterodimeric protein with double-stranded DNA end-binding activity that operates in the process of nonhomologous end joining. Ku is thought to target the DNA-dependent protein kinase (DNA-PK) complex to the DNA and, when DNA bound, can interact and activate the DNA-PK catalytic subunit (DNA-PKcs). We have carried out a 3' deletion analysis of Ku80, the larger subunit of Ku, and shown that the C-terminal 178 amino acid residues are dispensable for DNA end-binding activity but are required for efficient interaction of Ku with DNA-PKcs. Cells expressing Ku80 proteins that lack the terminal 178 residues have low DNA-PK activity, are radiation sensitive, and can recombine the signal junctions but not the coding junctions during V(D)J recombination. These cells have therefore acquired the phenotype of mouse SCID cells despite expressing DNA-PKcs protein, suggesting that an interaction between DNA-PKcs and Ku, involving the C-terminal region of Ku80, is required for DNA double-strand break rejoining and coding but not signal joint formation. To gain further insight into important domains in Ku80, we report a point mutational change in Ku80 in the defective xrs-2 cell line. This residue is conserved among species and lies outside of the previously reported Ku70-Ku80 interaction domain. The mutational change nonetheless abrogates the Ku70-Ku80 interaction and DNA end-binding activity.

At least two mechanisms of DNA double-strand break (DSB) repair operate in mammalian cells. Homologous recombination, which plays a major role in lower organisms, plays a less significant role in higher organisms. In contrast, the majority of DSBs in mammalian cells are rejoined by a mechanism, termed non-homologous end joining (NHEJ), that does not depend upon extensive regions of homology. This process is also used to rejoin site-specific DSBs introduced during V(D)J recombination. From the analysis of defective rodent mutants, four proteins (Ku70, Ku80, DNA-PKcs and Xrcc4) that function in this process in mammalian cells have been identified. DNA ligase IV is also strongly implicated since it associates strongly with XRCC4, and since DNA ligase IV-deficient yeast are defective in their ability to carry out NHEJ. In S. cerevisiae, Sir2p, Sir3p and Sir4p, three proteins required for transcriptional silencing, are also required for NHEJ. Additionally, the yeast mutants, xrs2, rad50 and mre11, which are defective in meiotic recombination, are also defective in NHEJ. Here I review the evidence implicating these proteins as functioning in NHEJ and discuss their properties and role in other pathways. The significance of DSB repair to clinical radiosensitivity and human disorders is also evaluated.

The DNA-dependent protein kinase is a mammalian protein complex composed of Ku70, Ku80, and DNA-PKcs subunits that has been implicated in DNA double-strand break repair and V(D)J recombination. Here, by gene targeting, we have constructed a mouse with a disruption in the kinase domain of DNA-PKcs, generating an animal model completely devoid of DNA-PK activity. Our results demonstrate that DNA-PK activity is required for coding but not for signal join formation in mice. Although our DNA-PKcs defective mice closely resemble Scid mice, they differ by having elevated numbers of CD4+CD8+ thymocytes. This suggests that the Scid mice may not represent a null phenotype and may retain some residual DNA-PKcs function.