Human RAD50 deficiency: Confirmation of a distinctive phenotype.

DNA double-strand breaks (DSBs) are highly toxic DNA lesions that can lead to chromosomal instability, loss of genes and cancer. The MRE11/RAD50/NBN (MRN) complex is keystone involved in signaling processes inducing the repair of DSB by, for example, in activating pathways leading to homologous recombination repair and nonhomologous end joining. Additionally, the MRN complex also plays an important role in the maintenance of telomeres and can act as a stabilizer at replication forks. Mutations in NBN and MRE11 are associated with Nijmegen breakage syndrome (NBS) and ataxia telangiectasia (AT)-like disorder, respectively. So far, only one single patient with biallelic loss of function variants in RAD50 has been reported presenting with features classified as NBS-like disorder. Here, we report a long-term follow-up of an unrelated patient with facial dysmorphisms, microcephaly, skeletal features, and short stature who is homozygous for a novel variant in RAD50. We could show that this variant, c.2524G > A in exon 15 of the RAD50 gene, induces aberrant splicing of RAD50 mRNA mainly leading to premature protein truncation and thereby, most likely, to loss of RAD50 function. Using patient-derived primary fibroblasts, we could show abnormal radioresistant DNA synthesis confirming pathogenicity of the identified variant. Immunoblotting experiments showed strongly reduced protein levels of RAD50 in the patient-derived fibroblasts and provided evidence for a markedly reduced radiation-induced AT-mutated signaling. Comparison with the previously reported case and with patients presenting with NBS confirms that RAD50 mutations lead to a similar, but distinctive phenotype.

The MRX Complex Ensures NHEJ Fidelity through Multiple Pathways Including Xrs2-FHA-Dependent Tel1 Activation.

Because DNA double-strand breaks (DSBs) are one of the most cytotoxic DNA lesions and often cause genomic instability, precise repair of DSBs is vital for the maintenance of genomic stability. Xrs2/Nbs1 is a multi-functional regulatory subunit of the Mre11-Rad50-Xrs2/Nbs1 (MRX/N) complex, and its function is critical for the primary step of DSB repair, whether by homologous recombination (HR) or non-homologous end joining. In human NBS1, mutations result truncation of the N-terminus region, which contains a forkhead-associated (FHA) domain, cause Nijmegen breakage syndrome. Here we show that the Xrs2 FHA domain of budding yeast is required both to suppress the imprecise repair of DSBs and to promote the robust activation of Tel1 in the DNA damage response pathway. The role of the Xrs2 FHA domain in Tel1 activation was independent of the Tel1-binding activity of the Xrs2 C terminus, which mediates Tel1 recruitment to DSB ends. Both the Xrs2 FHA domain and Tel1 were required for the timely removal of the Ku complex from DSB ends, which correlates with a reduced frequency of imprecise end-joining. Thus, the Xrs2 FHA domain and Tel1 kinase work in a coordinated manner to maintain DSB repair fidelity.

Nijmegen breakage syndrome fibroblasts expressing the C-terminal truncated NBN(p70) protein undergo p38/MK2-dependent premature senescence.

Fibroblasts from the progeroid Nijmegen breakage syndrome that express a truncated version of the nibrin protein (NBN(p70)) undergo premature senescence and have an enlarged morphology with high levels of senescence-associated ß-galactosidase, although they do not have F-actin stress fibres. Growth of these fibroblasts in the continuous presence of p38 inhibitors resulted in a large increase in replicative capacity and changed the cellular morphology so that the cells resembled young normal fibroblasts. A similar effect was seen using an inhibitor of the p38 downstream effector kinase MK2. These data suggest that NBN(p70) expressing cells undergo a degree of stress-induced replicative senescence via p38/MK2 activation, potentially due to increased telomere dysfunction, that may play a role in the progeroid features seen in this syndrome.

DNA damage and oxidative injury are associated with hypomyelination in the corpus callosum of newborn Nbn(CNS-del) mice.

Nijmegen breakage syndrome (NBS), caused by mutation of the Nbn gene, is a recessive genetic disorder characterized by immunodeficiency, elevated sensitivity to ionizing radiation, chromosomal instability, microcephaly, and high predisposition to malignancies. To explore the underlying molecular mechanisms of NBS microcephaly, Frappart et al. previously inactivated Nbn gene in the central nervous system (CNS) of mice by the nestin-Cre targeting gene system and generated Nbn(CNS-del) mice. Here we first report that Nbn gene inactivation induces the defective proliferation and enhanced apoptosis of the oligodendrocyte precursor cells (OPCs), contributing to the severe hypomyelination of the nerve fibers of the corpus callosum. Under conditions of DNA damage and oxidative stress, the distinct regulatory roles of ATM-Chk2 signaling and AKT/mTOR signaling are responsible for the defective proliferation and enhanced apoptosis of the Nbn-deficient OPCs. In addition, specific HDAC isoforms may play distinctive roles in regulating the myelination of the Nbn-deficient OPCs. However, brain-derived neurotrophic factor and nerve growth factor stimulation attenuates the oxidative stress and thereby increases the proliferation of the Nbn-deficient OPCs, which is accompanied by upregulation of the AKT/mTOR/P70S6K signaling pathway. Taken together, these findings demonstrate that DNA damage and oxidative stress resulting from Nbn gene inactivation are associated with hypomyelination of the nerve fibers of corpus callosum.

The NBN founder mutation-Evidence for a country specific difference in age at cancer manifestation.

BACKGROUND: Nijmegen breakage syndrome (NBS) is an autosomal-recessive chromosome instability disorder characterized by, among others, hypersensitivity to X-irradiation and an exceptionally high risk for lymphoid malignancy. The vast majority of NBS patients is homozygous for a common Slavic founder mutation, c.657del5, of the NBN gene, which is involved in the repair of DNA double-strand breaks (DSBs). The founder mutation also predisposes heterozygous carriers to cancer, apparently however, with a higher risk in the Czech Republic/Slovakia (CS) than in Poland. AIM: To examine whether the age of cancer manifestation and cancer death of NBN homozygotes is different between probands from CS and Poland. METHODS: The study is restricted to probands born until 1989, before replacement of the communist regime by a democratic system in CS and Poland, and a substantial transition of the health care systems. Moreover, all patients were recruited without knowledge of their genetic status since the NBN gene was not identified until 1998. RESULTS: Here, we show that cancer manifestation of NBN homozygotes is at a significantly earlier age in probands from CS than from Poland. This is explained by the difference in natural and medical radiation exposure, though within the permissible dosage. CONCLUSION: It is reasonable to assume that this finding also sheds light on the higher cancer risk of NBN heterozygotes in CS than in Poland. This has implications for genetic counseling and individualized medicine also of probands with other DNA repair defects.

Human RAD50 deficiency in a Nijmegen breakage syndrome-like disorder.

The MRE11/RAD50/NBN (MRN) complex plays a key role in recognizing and signaling DNA double-strand breaks (DSBs). Hypomorphic mutations in NBN (previously known as NBS1) and MRE11A give rise to the autosomal-recessive diseases Nijmegen breakage syndrome (NBS) and ataxia-telangiectasia-like disorder (ATLD), respectively. To date, no disease due to RAD50 deficiency has been described. Here, we report on a patient previously diagnosed as probably having NBS, with microcephaly, mental retardation, 'bird-like' face, and short stature. At variance with this diagnosis, she never had severe infections, had normal immunoglobulin levels, and did not develop lymphoid malignancy up to age 23 years. We found that she is compound heterozygous for mutations in the RAD50 gene that give rise to low levels of unstable RAD50 protein. Cells from the patient were characterized by chromosomal instability; radiosensitivity; failure to form DNA damage-induced MRN foci; and impaired radiation-induced activation of and downstream signaling through the ATM protein, which is defective in the human genetic disorder ataxia-telangiectasia. These cells were also impaired in G1/S cell-cycle-checkpoint activation and displayed radioresistant DNA synthesis and G2-phase accumulation. The defective cellular phenotype was rescued by wild-type RAD50. In conclusion, we have identified and characterized a patient with a RAD50 deficiency that results in a clinical phenotype that can be classified as an NBS-like disorder (NBSLD).

Cleavage of the BRCT tandem domains of nibrin by the 657del5 mutation affects the DNA damage response less than the Arg215Trp mutation.

The Nijmegen breakage syndrome (NBS) is a genetic disorder caused by mutations in NBN gene and characterized by chromosomal instability and hypersensitivity to ionizing radiations (IR). The N-terminus of nibrin (NBN) contains a tandem breast cancer 1 (BRCA1) carboxy-terminal (BRCT) domain that represents one of the major mediators of phosphorylation-dependent protein-protein interactions in processes related to cell cycle checkpoint and DNA repair functions. Patients with NBS compound heterozygous for the 657del5 hypomorphic mutation and for the Arg215Trp missense mutation (corresponding to the 643C>T gene mutation) display a clinical phenotype more severe than that of patients homozygous for the 657del5 mutation. Here, we show that both the 657del5 and Arg215Trp mutations, occurring within the tandem BRCT domains of NBN, although not altering the assembly of the MRE11/RAD50/NBN (MRN) complex, affect the MRE11 IR-induced nuclear foci (IRIF) formation and the DNA double-strand break (DSB) signaling via the phosphorylation of both ataxia-telangiectasia-mutated (ATM) kinase and ATM downstream targets (e.g., SMC1 and p53). Remarkably, data obtained indicate that the cleavage of the BRCT tandem domains of NBN by the 657del5 mutation affects the DNA damage response less than the Arg215Trp mutation. Indeed, the 70-kDa NBN fragment, arising from the 657del5 mutation, maintains the capability to interact with MRE11 and γ-H2AX and to form IRIF. Altogether, the role of the tandem BRCT domains of NBN in the localization of the MRN complex at the DNA DSB and in the activation of the damage response is highlighted.

The defect in humoral immunity in patients with Nijmegen breakage syndrome is explained by defects in peripheral B lymphocyte maturation.

Patients with an immunodeficiency in the course of Nijmegen breakage syndrome (NBS) that is caused by mutations in the NBN/NBS1 gene are prone to recurrent infections and malignancies, due to a defective DNA double-strand breaks repair mechanism. Four-color flow cytometry was used to analyze changes in B lymphocyte subsets reflecting the most important stages of peripheral B cell maturation. It was demonstrated that the humoral immune defect observed in NBS patients was caused by reduced numbers of B lymphocytes, but also by their aberrant maturation. Reduced relative and absolute counts of naïve and memory B cells were accompanied by a significant accumulation of the natural effector B lymphocytes. The elevated proportion of IgM-only memory and reduced proportion of IgM-negative cells within the memory B cell pool suggests that there is class-switch recombination defect in this population of cells in NBS patients, resulting in inadequate production of immunoglobulins. Because of the reduced T-cell counts, the T-cell dependent antigen response is severely impaired resulting in a lower frequency of memory B-cells. The T-cell independent B-cell differentiation pathway seems less affected. The reduced IgG and IgA levels in patients with NBS are caused both by ineffective class switch, at least due to poor T cell help, and low number of memory B cells. This study illustrates that the NBN gene product nibrin plays an important role at different levels in the B-cell system.

Loss of juxtaposition of RAG-induced immunoglobulin DNA ends is implicated in the precursor B-cell differentiation defect in NBS patients.

The Nijmegen breakage syndrome (NBS) is a rare inherited condition, characterized by microcephaly, radiation hypersensitivity, chromosomal instability, an increased incidence of (mostly) lymphoid malignancies, and immunodeficiency. NBS is caused by hypomorphic mutations in the NBN gene (8q21). The NBN protein is a subunit of the MRN (Mre11-Rad50-NBN) nuclear protein complex, which associates with double-strand breaks. The immunodeficiency in NBS patients can partly be explained by strongly reduced absolute numbers of B lymphocytes and T lymphocytes. We show that NBS patients have a disturbed precursor B-cell differentiation pattern and significant disturbances in the resolution of recombination activating gene-induced IGH breaks. However, the composition of the junctional regions as well as the gene segment usage of the reduced number of successful immunoglobulin gene rearrangements were highly similar to healthy controls. This indicates that the NBN defect leads to a quantitative defect in V(D)J recombination through loss of juxtaposition of recombination activating gene-induced DNA ends. The resulting reduction in bone marrow B-cell efflux appeared to be partly compensated by significantly increased proliferation of mature B cells. Based on these observations, we conclude that the quantitative defect will affect the B-cell receptor repertoire, thus contributing to the observed immunodeficiency in NBS patients.

Impact of heterozygous c.657-661del, p.I171V and p.R215W mutations in NBN on nibrin functions.

Nibrin, product of the NBN gene, together with MRE11 and RAD50 is involved in DNA double-strand breaks (DSBs) sensing and repair, induction of apoptosis and cell cycle control. Biallelic NBN mutations cause the Nijmegen breakage syndrome, a chromosomal instability disorder characterised by, among other things, radiosensitivity, immunodeficiency and an increased cancer risk. Several studies have shown an association of heterozygous c.657-661del, p.I171V and p.R215W mutations in the NBN gene with a variety of malignancies but the data are controversial. Little is known, however, whether and to what extent do these mutations in heterozygous state affect nibrin functions. We examined frequency of chromatid breaks, DSB repair, defects in S-phase checkpoint and radiosensitivity in X-ray-irradiated cells from control individuals, NBS patients and heterozygous carriers of the c.657-661del, p.I171V and p.R215W mutations. While cells homozygous for c.657-661del displayed a significantly increased number of chromatid breaks and residual γ-H2AX foci, as well as abrogation of the intra-S-phase checkpoint following irradiation, which resulted in increased radiosensitivity, cells with heterozygous c.657-661del, p.I171V and p.R215W mutations behaved similarly to control cells. Significant differences in the frequency of spontaneous and ionising radiation-induced chromatid breaks and the level of persistent γ-H2AX foci were observed when comparing control and mutant cells heterozygous for c.657-661del. However, it is still possible that heterozygous NBN mutations may contribute to cancer development.

Promising therapy results for lymphoid malignancies in children with chromosomal breakage syndromes (Ataxia teleangiectasia or Nijmegen-breakage syndrome): a retrospective survey.

Children with chromosomal instability syndromes have an increased risk of developing lymphoma and leukaemia. The treatment of these malignancies is hampered by therapy-associated toxicity and infectious complications. This retrospective analysis evaluated the therapy outcome of 38 children with Ataxia teleangiectasia or Nijmegen-breakage syndrome with acute lymphoblastic leukaemia (ALL, n = 9), Non-Hodgkin lymphoma (NHL, n = 28) and Hodgkin lymphoma (HL, n = 1). All patients with NHL or ALL were treated in accordance to Berlin-Frankfurt-Münster (BFM)- or Co-operative study group for childhood ALL (CoALL)-oriented chemotherapy schedules. 22 patients received significantly reduced-intensity chemotherapy. After a median follow-up of 3·7 years the 10-year overall survival was 58%. Dosage-reduction of chemotherapeutic drugs seemed to have no disadvantages and reduced toxic side effects. On the other hand, reduced-intensity chemotherapy did not prevent second malignancies, which occurred in ten patients with a 10-year incidence of 25%. After individual treatment approaches three of these patients with second malignancies were in complete clinical remission for more than 5 years. We conclude that BFM- or CoALL-oriented chemotherapy is effective and can be administered in children with AT or NBS. Moreover, we show that even second lymphoid malignancies can successfully be treated in these patients.

Nijmegen breakage syndrome (NBS) as a risk factor for CNS involvement in childhood acute lymphoblastic leukemia.

Central nervous system (CNS) involvement is an independent risk factor for poor event-free survival and relapse confined to the CNS. Knock-out mice deprived of RAG2, the protein involved in DNA repair, developed leukemic infiltration within leptomeninges. Therefore, we hypothesized that DNA repair deficiencies in humans, such as Nijmegen breakage syndrome (NBS), may constitute a risk factor for CNS dissemination of acute lymphoblastic leukemia (ALL). Having analyzed the incidence of CNS2/CNS3 status at diagnosis of ALL in two independent cohorts from the Polish Pediatric Leukemia/Lymphoma Study Group, we noticed that among children with NBS CNS involvement was significantly frequent.

Clinical variability and novel mutations in the NHEJ1 gene in patients with a Nijmegen breakage syndrome-like phenotype.

We have previously shown that mutations in the genes encoding DNA Ligase IV (LIGIV) and RAD50, involved in DNA repair by nonhomologous-end joining (NHEJ) and homologous recombination, respectively, lead to clinical and cellular features similar to those of Nijmegen Breakage Syndrome (NBS). Very recently, a new member of the NHEJ repair pathway, NHEJ1, was discovered, and mutations in patients with features resembling NBS were described. Here we report on five patients from four families of different ethnic origin with the NBS-like phenotype. Sequence analysis of the NHEJ1 gene in a patient of Spanish and in a patient of Turkish origin identified homozygous, previously reported mutations, c.168C>G (p.Arg57Gly) and c.532C>T (p.Arg178Ter), respectively. Two novel, paternally inherited truncating mutations, c.495dupA (p.Asp166ArgfsTer20) and c.526C>T (p.Arg176Ter) and two novel, maternal genomic deletions of 1.9 and 6.9 kb of the NHEJ1 gene, were found in a compound heterozygous state in two siblings of German origin and in one Malaysian patient, respectively. Our findings confirm that patients with NBS-like phenotypes may have mutations in the NHEJ1 gene including multiexon deletions, and show that considerable clinical variability could be observed even within the same family.

Nijmegen breakage syndrome protein (NBN) causes resistance to methylating anticancer drugs such as temozolomide.

Methylating agents are first-line therapeutics for gliomas and malignant melanomas. They attack DNA at various sites, and both O(6)-methylguanine and N-methylated base adducts contribute to the killing response. The mechanism of cellular defense against these agents primarily involves O(6)-methylguanine-DNA methyltransferase (MGMT) and base excision repair (BER). Here, we determined whether a key protein involved in DNA double-strand break (DSB) recognition and signaling, nibrin (NBN alias NBS-1), plays a role in the cellular defense against methylating agents. Comparing NBN mutated fibroblasts and lymphoblastoid cells from patients suffering from Nijmegen breakage syndrome, we show that NBN mutants are clearly more sensitive to N-methyl-N'-nitro-N-nitrosoguanidine and temozolomide than the corresponding wild-type cells. Hypersensitivity was due to the induction of both apoptosis and necrosis. The mismatch repair proteins MSH2, MSH6, MLH1, and PMS2 were expressed at a similar level in the cell lines and BER was not affected by NBN mutation. Because MGMT expression abrogated the hypersensitivity of NBN mutated cells, we conclude that O(6)-methylguanine-derived lesions are responsible for triggering the response. Down-regulation of NBN in melanoma cells by small interfering RNA rendered them more sensitive to temozolomide, suggesting that NBN is a novel modulator of temozolomide sensitivity. Because NBN is part of the MRN complex, which recognizes DSBs, the data strongly indicate that MRN is critically involved in DSB processing after O(6)-methylguanine induction. The data provide first evidence that NBN is involved in the cellular defense against O(6)-methylguanine-inducing agents such as temozolomide and identify NBN as a critical target of methylating anticancer drug resistance.

Chronic noninfectious necrotizing granulomas in a child with Nijmegen breakage syndrome.

Nijmegen breakage syndrome (NBS) is a chromosomal breakage disorder with characteristic physical features, chromosomal instability, and combined immunodeficiency. It is closely related to other chromosomal breakage disorders like ataxia telangiectasia. Noninfectious granulomatous inflammation refractory to treatment is a relatively common feature in ataxia telangiectasia. Herein we report a patient with NBS who developed chronic refractory necrotizing granulomatous ulcerations and review the pathophysiology of NBS and noninfectious granulomas in primary immunodeficiency syndromes.

Association of Nijmegen Breakage Syndrome 1 Genotypes With Bladder Cancer Risk.

BACKGROUND/AIM: We aimed to examine the association of the genotypes of Nijmegen breakage syndrome 1 (NBS1), a critical gene in DNA double strand break repair machinery, with bladder cancer risk in Taiwan. MATERIALS AND METHODS: NBS1 rs1805794 genotypes among 375 bladder cancer patients and 375 non-cancer healthy controls were determined via the polymerase chain reaction-restriction fragment length polymorphism methodology and their association with bladder cancer risk were evaluated. RESULTS: The results showed that the percentages of GG, CG and CC of NBS1 rs1805794 genotypes were 45.4%, 43.7% and 10.9% in the bladder cancer patient group and 47.2%, 43.2% and 9.6% in the non-cancer control group, respectively (p for trend=0.7873). The analysis of allelic frequency distributions showed that the variant C allele of NBS1 rs1805794 does not contribute to an increased bladder cancer susceptibility (p=0.5066). CONCLUSION: The genotypes of NBS1 rs1805794 are not closely associated with personal susceptibility to bladder cancer.

Telomere attrition and dysfunction: a potential trigger of the progeroid phenotype in nijmegen breakage syndrome.

BACKGROUND: Nibrin, as part of the NBN/MRE11/RAD50 complex, is mutated in Nijmegen breakage syndrome (NBS), which leads to impaired DNA damage response and lymphoid malignancy. RESULTS: Telomere length (TL) was markedly reduced in homozygous patients (and comparably so in all chromosomes) by ~40% (qPCR) and was slightly reduced in NBS heterozygotes older than 30 years (~25% in qPCR), in accordance with the respective cancer rates. Humanized cancer-free NBS mice had normal TL. Telomere elongation was inducible by telomerase and/or alternative telomere lengthening but was associated with abnormal expression of telomeric genes involved in aging and/or cell growth. Lymphoblastoid cells from NBS patients with long survival times (>12 years) displayed the shortest telomeres and low caspase 7 activity. CONCLUSIONS: NBS is a secondary telomeropathy. The two-edged sword of telomere attrition enhances the cancer-prone situation in NBS but can also lead to a relatively stable cellular phenotype in tumor survivors. Results suggest a modular model for progeroid syndromes with abnormal expression of telomeric genes as a molecular basis. METHODS: We studied TL and function in 38 homozygous individuals, 27 heterozygotes, one homozygous fetus, six NBS lymphoblastoid cell lines, and humanized NBS mice, all with the same founder NBN mutation: c.657_661del5.

Fertility defects revealing germline biallelic nonsense NBN mutations.

Biallelic mutations in the NBN/NBS1 gene are the cause of Nijmegen breakage syndrome (NBS), a severe pediatric disease characterized by dysmorphy with a bird-like face, microcephaly, growth retardation, immune deficiency, and proneness to cancer. We here report two adult siblings that are compound heterozygotes for two previously unreported NBN nonsense mutations. These patients presented with the unique clinical symptom of fertility defects. Contrasting with the absence of any developmental abnormality, biological analyses revealed defects similar to those observed in NBS patients, including chromosomal instability, cellular hyperradiosensitivity and checkpoint defects as measured by radioresistant DNA synthesis (RDS). NBN mutations should thus be considered a new cause of infertility, and should be searched for if associated with the biological abnormalities of NBS.

DNA double-strand break and chromosomal rejoining defects with misrejoining in Nijmegen breakage syndrome cells.

NBS1-deficient cells exhibit pronounced radiosensitivity and defects in chromosome integrity after ionizing radiation (IR) exposure, yet show only a minor defect in DNA double-strand break (DSB) rejoining, leaving an as yet unresolved enigma as to the nature of the radiosensitivity of these cells. To further investigate the relationship between radiosensitivity, DSB repair, and chromosome stability, we have compared cytological and molecular assays of DSB misrejoining and repair in NBS1-defective, wild type, and NBS1-complemented cells after IR damage. Our findings suggest a subtle defect in overall DSB rejoining in NBS1-defective cells and uniquely also reveal reduced ability of NBS1-defective cells to rejoin correct ends of DSBs. In agreement with published results, one of two different NBS1-defective cell lines showed a slight defect in overall rejoining of DSBs compared to its complemented counterpart, whereas another NBS line did not show any difference from wild type cells. Significant defects in the correct rejoining of DSBs compared to their respective controls were observed for both NBS1-defective lines. The defect in DSB rejoining and the increased misrejoining detected at the molecular level were also reflected in higher levels of fragments and translocations, respectively, at the chromosomal level. This work provides both molecular and cytological evidence that NBS1-deficient cells have defects in DSB processing and reveals that these molecular events can be manifest cytologically.

Growth of persistent foci of DNA damage checkpoint factors is essential for amplification of G1 checkpoint signaling.

Several DNA damage checkpoint factors form nuclear foci in response to ionizing radiation (IR). Although the number of the initial foci decreases concomitantly with DNA double-strand break repair, some fraction of foci persists. To date, the physiological role of the persistent foci has been poorly understood. Here we examined foci of Ser1981-phosphorylated ATM in normal human diploid cells exposed to 1Gy of X-rays. While the initial foci size was approximately 0.6microm, the one or two of persistent focus (foci) grew, whose diameter reached 1.6microm or more in diameter at 24h after IR. All of the grown persistent foci of phosphorylated ATM colocalized with the persistent foci of Ser139-phosphorylated histone H2AX, MDC1, 53BP1, and NBS1, which also grew similarly. When G0-synchronized normal human cells were released immediately after 1Gy of X-rays and incubated for 24h, the grown large phosphorylated ATM foci (> or =1.6microm) were rarely (av. 0.9%) observed in S phase cells, while smaller foci (<1.6microm) were frequently (av. 45.9%) found. We observed significant phosphorylation of p53 at Ser15 in cells with a single grown phosphorylated ATM focus. Furthermore, persistent inhibition of foci growth of phosphorylated ATM by an ATM inhibitor, KU55933, completely abrogated p53 phosphorylation. Defective growth of the persistent IR-induced foci was observed in primary fibroblasts derived from ataxia-telangiectasia (AT) and Nijmegen breakage syndrome (NBS) patients, which were abnormal in IR-induced G1 checkpoint. These results indicate that the growth of the persistent foci of the DNA damage checkpoint factors plays a pivotal role in G1 arrest, which amplifies G1 checkpoint signals sufficiently for phosphorylating p53 in cells with a limited number of remaining foci.