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Human Molecular Genetics Advance Access originally published online on December 1, 2004
Human Molecular Genetics 2005 14(2):307-318; doi:10.1093/hmg/ddi027
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Human Molecular Genetics, Vol. 14, No. 2 © Oxford University Press 2005; all rights reserved

Identification and functional consequences of a novel MRE11 mutation affecting 10 Saudi Arabian patients with the ataxia telangiectasia-like disorder

Marie Fernet1,{dagger},{ddagger}, Moez Gribaa2,3,{dagger}, Mustafa A.M. Salih4, Mohamed Zein Seidahmed5, Janet Hall1,* and Michel Koenig2

1DNA Repair Team, International Agency for Research on Cancer, 150 cours Albert Thomas, Lyon 69372, France, 2Institut de Génétique et de Biologie Moléculaire et Cellulaire (CNRS/INSERM/ULP), Illkirch - Strasbourg 67404, France, 3Service de Génétique et de Cytogénétique, Hôpital Universitaire Farhat Hached, Sousse, Tunisia, 4Division of Paediatric Neurology, Department of Paediatrics, College of Medicine, King Saud University, Riyadh 11461, Saudi Arabia and 5Department of Paediatrics, Security Forces Hospital, Riyadh 11481, Saudi Arabia

* To whom correspondence should be addressed. Tel: +33 472738596; Fax: +33 472738322; Email: hall{at}iarc.fr

Received September 14, 2004; Accepted November 16, 2004


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Ten new patients with ataxia telangiectasia-like disorder (ATLD) from three unrelated Saudi Arabian families have been identified aged 5–37 representing the largest cohort of ATLD patients ever identified. They presented with an early-onset, slowly progressive, ataxia plus ocular apraxia phenotype with an absence of tumor development, even in the oldest patient. Extra-neurological features such as telangiectasia, raised alpha-fetoprotein and reduced immunoglobulin levels were absent. No translocations were found in the two investigated patients, and the presence of microcephaly was noted in four out of eight ascertained patients. All patients are homozygous for a novel missense mutation (630G->C, W210C) of the MRE11 gene. The cellular consequences of this amino acid change, localized in the nuclease domain of the Mre11 protein, have been determined in fibroblast cultures established from two individuals. They showed high constitutive levels of Mre11 and Rad50 proteins compared with cells from normal individuals but a very low level of the Nbs1 protein. After exposure to ionizing radiation, a dose-dependent defect in ataxia telangiectasia mutated (ATM)-serine 1981, p53-serine 15 and Chk2 phosphorylation, and p53 stabilization were noted, together with a failure to form Mre11 foci and enhanced radiation sensitivity. Formation of {gamma}H2AX foci was similar to that seen in normal fibroblasts under the experimental conditions examined. These results emphasize the importance of functional interactions among the three proteins of the Mre11–Rad50–Nbs1 complex and lend support to a role of this complex as a sensor of DNA double-strand breaks, acting upstream of ATM.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The presence of DNA double-strand breaks (DSBs) in mammalian cells activates a complex array of responses that result in cell cycle checkpoints activation, DNA repair and the onset of apoptosis. Following exposure to ionizing radiation (IR), the rapid activation of these signaling cascades requires the presence of the ataxia telangiectasia mutated (ATM) protein and the Mre11–Rad50–Nbs1 (MRN) complex (1Go–3Go).

The ATM protein is held inactive in undamaged cells as a dimer or high-order multimer, with the kinase domain bound to a region surrounding serine 1981. In response to the formation of DNA DSBs, the autophosphorylation of this serine results in dimer dissociation and activation of the kinase activity of ATM (1Go). The subsequent phosphorylation of target proteins, including p53, Chk1, Chk2, HDM2, SMC1, Nbs1, FANCD2, BRCA1, 53BP1, Mre11 and RPA32, results in the activation of cell cycle checkpoints and DNA repair. The measurement of these events in protein extracts from treated cells can be used to monitor ATM activity. For instance, the phosphorylation of p53 on serine 15 and Chk2 on threonine 68, two crucial targets in respect to the regulation of the G1/S checkpoint, is compromised in ATM deficient cells (2Go,4Go–6Go).

Recent evidence suggests that the MRN complex also functions as a lesion-specific sensor, acting upstream of the ATM protein in damage signaling, and that the Nbs1 protein, itself an ATM substrate, serves as an adaptor in the phosphorylation of certain ATM substrates such as Chk2 and SMC1 (7Go–13Go). When cells are treated with IR, one of the earliest events following the formation of DSBs is the phosphorylation of the histone H2A isoform, H2AX, by ATM, which initiates the formation of foci (14Go,15Go). These consist of a complex array of proteins localized to the region surrounding the DSB in a dynamically organized and timely manner (16Go). Foci can be observed from 10 min after exposure to IR and contain H2AX, ATM, MRN proteins and mediator proteins such as BRCA1, 53BP1 and MDC1 (17Go–21Go). They will persist until at least 24 h after treatment, with a maximal induction of MRN foci 8 h post-irradiation (3Go,22Go).

Mutations in ATM, MRE11 or NBS1 genes are associated with rare syndromes, all of which are characterized by an increased sensitivity to IR (23Go). The ATM gene is mutated in patients with the disorder ataxia telangiectasia (AT), characterized also by cerebellar degeneration, immunodeficiency, chromosomal instability and cancer predisposition (24Go,25Go). Hypomorphic mutations in the NBS1 locus lead to Nijmegen breakage syndrome (NBS), characterized also by microcephaly, mental deficiency, immunodeficiency, chromosomal instability and cancer predisposition (23Go,26Go). MRE11 mutations are the underlying cause of the AT-like disorder (ATLD), a very rare slowly progressive variant of AT with, at present, six known cases: four in the UK and two in Italy. The clinical features of patients with ATLD are very similar to those with AT, with ATLD patients showing progressive cerebellar ataxia but not telangiectasia. However, the neurological features have a later onset and slower progression (20Go,27Go–29Go).

To date, two truncating and two missense mutations have been reported in ATLD patients. Cells from these patients express variable levels of Mre11 protein, which is either truncated or full-length and mutant. Interestingly, this alteration in Mre11 protein level is associated with a reduction in levels of Nbs1 and Rad50 proteins (20Go,27Go,28Go). Quantitative differences in the cellular response to IR in terms of cell survival, the activation of DNA damage signaling pathways and foci formation exist between ATLD and NBS cells, with a cellular phenotype less severe than that observed in cells carrying ATM mutations (13Go,20Go,27Go).

Here, we describe 10 new patients from three distinct Saudi Arabian families presenting with an early onset, slowly progressive, ataxia plus ocular apraxia phenotype with an absence of immune deficiency and tumor development, even at advanced age. These patients represent the largest cohort of ATLD patients ever identified. All patients are homozygous for a novel missense mutation (630G->C, W210C) of the MRE11 gene. In order to examine the phenotypic consequences of this mutation, cells from two patients were characterized in terms of Mre11, Nbs1 and Rad50 protein levels and their cellular response to ionizing radiation. The endpoints studied were cell survival, cell cycle progression, ATM and H2AX activation, Mre11 and Nbs1 foci formation, p53 induction and phosphorylation of p53-ser15 and Chk2, following treatment with variable doses of irradiation.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Identification of MRE11 mutation
Three hundred and ninety markers of the ABI PRISM linkage mapping set were genotyped with family 1, from Saudi Arabia. Given the close consanguinity of the parents and the close spacing of the markers, we selected the regions for which at least one marker was homozygous in all four affected individuals and heterozygous in the healthy sibling available at the time of the study. We identified only one such region, on chromosome 11q22–q23, in which six consecutive markers (from D11S937 to D11S925, 39 cM) were homozygous in all patients. The study of a dense set of GA/TG microsatellite markers from this region and three additional available siblings subsequently confirmed linkage to the 11q locus, because the four patients were homozygous for 14 consecutive markers and the healthy siblings inherited at least one different haplotype (Fig. 1A). LOD score calculation, including the consanguinity loop (30Go,31Go), gave a value of 2.9 at a recombination fraction {theta} of 0, in favor of linkage. We then studied 30 additional consanguineous families with recessive non-Friedreich, non-AT ataxia, from various geographic origins. One additional family (family 2, also from Saudi Arabia) showed linkage to the same region, with the three patients homozygous for five to eight consecutive markers out of the 11 tested and the three healthy siblings inheriting one different haplotype (Fig. 1B). LOD score calculations for family 2 gave a value of 2.77 at a recombination fraction {theta} of 0, in favor of linkage. The clinical presentation of patients in family 2 was very similar to that of patients in family 1, with the exception of brisk deep tendon reflexes in the former and reduced reflexes in the latter (Table 1). Two known recessive ataxia genes, ATM and MRE11, are located in the linked interval of families 1 and 2. Because both families were missing essential features of AT, such as elevated alpha-fetoprotein (AFP) and immune deficiency, we focused our efforts on MRE11 and identified in both families the same G to C transversion at position 630 (630G->C in exon 7), which results in the missense change W210C (change of tryptophan to cysteine). No other nucleotide change was identified in the coding sequences or flanking intronic sequences of MRE11 of families 1 and 2. This mutation was found by direct sequencing of exon 7 in one of two additional Saudi Arabian families examined with ataxia plus ocular apraxia (family 3). The segregation of the 630G->C change in family 3 (homozygosity in the three patients and heterozygosity or absence in the four healthy siblings) gave a LOD score of 2.9, in favor of linkage of this nucleotide change with ataxia in this family. The clinical features of the patients from all three families are summarized in Table 1. The 630G->C change was not found in 192 unrelated control individuals from Arabic or European origin. Tryptophan at position 210 is a highly conserved residue in the Mre11 protein, being invariable from yeast to mammals (Fig. 2), and its replacement by the small polar residue cysteine is likely to affect the structure or function of the protein. In addition, this residue lies between motifs III and IV of the N-terminal nuclease domain of the Mre11 protein, a region where another missense mutation (N117S) had been identified in ATLD patients (20Go). All these data allow the diagnosis of ATLD, caused by the W210C mutation, to be established for the patients from families 1, 2 and 3. In order to gain additional insight on the pathogenicity of this mutation, the biochemical pathway of the Mre11 protein was investigated in the fibroblast cell line of one patient from families 1 and 2 (lines ATLD7 and ATLD8, respectively).



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  		Figure 1. Genotyping results of families 1 (A) and 2 (B) for the chromosome 11q22–q23 region. Patient numbers are as in Table 1; patients 3 and 4 of family 1 are monozygotic twins. D11S number of markers is indicated on the left and is followed by marker position on the genetic linkage map (in cM in brackets). Markers are organized from top to bottom in the centromeric to telomeric order. Parental haplotypes linked with the disease are boxed. Brackets around parental allele sizes indicate that they were inferred from the children genotypes. Dash (–), not tested. The region of homozygosity by descent is highlighted in gray.

 

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  		Table 1. Clinical and biochemical features of the ATLD patients
 


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  		Figure 2. Alignment of MRE11 sequences from different species with the human stretch extending from amino acids 197–266. The mutated residue is indicated in bold and the mutant residue is indicated on top of the human sequence. The phosphoesterase motif IV of the nuclease domain is overlined.

 
Basal levels of Mre11, Nbs1 and Rad50 proteins
The basal levels of the MRN complex proteins in fibroblast cultures from two patients of the newly identified ATLD families, in comparison with normal, AT and ATLD2 cells, were studied using western blots (Fig. 3). The protein extracts from ATLD2 cells, carrying a homozygous truncating mutation in MRE11 gene, showed a null level of Mre11 and very low levels of both Nbs1 and Rad50 proteins as previously reported (20Go).



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  		Figure 3. Basal levels of MRN proteins. Whole-cell protein extracts were made from primary fibroblast cell cultures derived from normal individuals (1BR3 [PDB] and 251BR), an AT patient (93RD346), the ATLD2 patient and two new ATLD patients (ATLD7 and ATLD8) and separated by SDS–PAGE. Western blot analysis was performed using antibodies directed against Rad50, Mre11 and Nbs1 proteins. The western blot was additionally probed for actin to standardize for protein loading.

 
Quantification by densitometric analysis of at least two extracts for each cell line showed that ATLD7 and ATLD8 cells expressed higher levels of Mre11 (P<0.001) and Rad50 proteins than the four normal cells (P<0.001 and P=0.0016 for ATLD7 and ATLD8, respectively), but a lower Nbs1 protein level (P<0.001). Differences were noted between ATLD7 and ATLD8 cells, with a lower level of Rad50 (P=0.049) and a significantly lower level of Nbs1 (P=0.0012) in ATLD8. The level of the Nbs1 protein was similar in ATLD8 and ATLD2 cells (P=0.55).

Cell survival
The relative cell survival of the cell lines studied after exposure to 1, 2 and 4 Gy is shown in Figure 4. Statistical analysis showed no differences between the two new ATLD cell cultures and the normal cells after exposure to 1 Gy (P=0.66 and P=0.2 for ATLD7 and ATLD8, respectively) but a lower cell survival for ATLD8 after exposure to 2 Gy (ATLD8 versus normals, P=0.015; ATLD7 versus normals, P=0.105). After exposure to 4 Gy, both new ATLD cell cultures had a lower cell survival than the normal cell cultures (P<0.001 and P=0.002 for ATLD7 and ATLD8 versus normals, respectively). At all doses examined, ATLD7 and ATLD8 were less radiosensitive than the NBS and AT cells examined. The cell survival of ATLD7 cells could not be distinguished from that of ATLD2 cells at any of the treatment doses; however, ATLD8 showed a slightly higher cell survival following a 4 Gy irradiation (P=0.03 for ATLD8 versus ATLD2).



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  		Figure 4. Radiation cell survival curves. The viable cells were counted by trypan blue exclusion 12 days after exposure to gamma radiation. The number of cell in non-irradiated flasks was considered as 100% of cell survival. Relative cell survival of cells from new ATLD patients (ATLD7 and ATLD8) was compared with cells from three normal individuals (1BR3, 85RD469 and 251BR), an AT patient (93RD346), the ATLD patient (ATLD2) and an NBS patient (CZD82H). Mean values±standard deviation were established based on the results from at least two independent experiments in duplicate, on each cell culture.

 
Cell cycle progression
Progression of the cells into the cell cycle after irradiation was studied 14 and 24 h after exposure to 3 Gy. The percentages of cells in G0/G1 and G2/M phases are summarized in Table 2. All cell cultures examined exhibited a radiation-induced arrest in G1 phase that was more accentuated after 24 h, as seen in treated normal cells. However, ATLD8 and ATLD2 cells showed fewer cells in G0/G1 and more cells in G2/M compared with normal cells, but these differences were not statistically significance. It should be noted that percentages of ATLD7 and ATLD8 cells in each phase are significantly different (P=0.0495 for both time points). These results suggest that the ATLD7 and ATLD8 cells show no major cell cycle checkpoint defect 14 and 24 h after exposure to 3 Gy.


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  		Table 2. Cell cycle progression after exposure to 3 Gy of gamma-radiation
 
Ionizing radiation-induced foci
After exposure to ionizing radiation, ATM protein is activated by auto-phosphorylation of ATM dimers on serine 1981 and localizes at DNA DSB sites, forming foci. The immunostaining of phosphorylated ATM protein in cells without irradiation and 30 min after exposure to 0.5 or 3 Gy is shown in Figure 5. After an irradiation with 3 Gy, some foci are formed in normal, ATLD7 and ATLD8 and ATLD2 cell cultures but to a lesser extent in all ATLD cells, compared with normal cells. No foci were observed in AT cells. Following lower dose irradiation (0.5 Gy), foci were detectable only in normal cells. These results suggest that the defect in ATM activation in the ATLD cells is dose dependent.



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  		Figure 5. Phosphorylated ATM and H2AX foci induced by IR. Foci were detected using antibodies directed against serine 1981 of ATM protein or serine 139 of H2AX protein in cells from normal individuals (N is representative of all the normal cells examined), ATLD7 and ATLD8 cells (ATLD is representative of both) and cells from NBS (CZD82H), ATLD2 and AT (93RD346) patients. Cells were fixed and stained 30 min after irradiation with 0.5 or 3 Gy or mock-irradiation, as described in Materials and Methods.

 
In the presence of DNA DSBs, H2AX protein is rapidly phosphorylated on serine 139 by ATM and DNA-PK. Thirty minutes after exposure to 3 Gy, formation of {gamma}H2AX foci was observed in normal cells, as shown in Figure 5. The ATLD7, ATLD8 and ATLD2 cell cultures exhibited the same pattern of {gamma}H2AX foci as normal cell cultures, foci were also formed in AT and NBS cells but were smaller and more numerous than in normal cells. Formation of {gamma}H2AX foci was also observed in ATLD cells after 0.5 Gy irradiation (data not shown).

Mre11 and Nbs1 ionizing radiation-induced foci (IRIF) were studied 8 h after exposure to 10 Gy, these experimental conditions corresponding to type III Mre11 foci, which have been described as being dependent upon the process of DSB repair (32Go) and in which Nbs1 is co-localized. No formation of Mre11 or Nbs1 foci was observed in ATLD7 and ATLD8 cells or in ATLD2 and NBS cells (Fig. 6). These results suggest that the Mre11 and Nbs1 proteins detected by western blotting in protein extracts from these two new ATLD cell cultures are functionally deficient.



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  		Figure 6. Ionizing radiation-induced Mre11 and Nbs1 foci. Cells were collected 8 h after treatment with 10 Gy or without treatment. Cell cultures are those described in Figure 5.

 
p53 and Chk2 phosphorylation and p53 induction
After exposure to ionizing radiation, the p53 protein is phosphorylated on serine 15, partly by the ATM kinase, leading to its stabilization. As shown by western blot analysis (Fig. 7), ATLD7 and ATLD8 cells exhibited a reduced level of this p53 phosphorylation after exposure to 0.5 and 3 Gy, which was more pronounced in ATLD8 cells. ATLD cells also showed a deficient p53 induction after a 0.5 Gy irradiation and no Chk2 phosphorylation, as measured by the protein shift, after 3 or 10 Gy. The ATLD7 and ATLD8 cells exhibited 1.13±0.27-fold and 1.35±0.19-fold increase in p53 levels, respectively, 1 h after a 0.5 Gy irradiation, whereas the average increase was 2.25±0.23 for the normal cells. A defect was also observed 2 h after irradiation, with a 2.42±0.35-fold increase in the normal cells and only a 1.54±0.71-fold and 1.2±0.27-fold increase in the ATLD7 and ATLD8 cells, respectively. As previously described, the AT cells studied here showed no p53 and Chk2 phosphorylation and no p53 stabilization following these treatment schedules. However, no defect in p53 induction was observed in ATLD7 and ATLD8 cell cultures after treatment with 3 Gy (data not shown). These results suggest a deficient radiation-induced ATM kinase activity in ATLD7 and ATLD8 cells. The dose dependence in the cellular response can be explained by the activity of kinases other than ATM, such as ATR (ATM and Rad3 related) and DNA-PK, which phosphorylate many of the same proteins phosphorylated by ATM, particularly following high-radiation doses.



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  		Figure 7. Western blot analysis of radiation-induced p53 and Chk2 protein modifications in two normal individuals (1BR3 [PDB] and 251BR), the two new ATLD patients (ATLD7 and ATLD8), the ATLD2 and AT (93RD346) patients. Ku80 immunodetection was performed on each gel to correct for variations in protein loading. (A) Phosphorylation of p53 on serine 15 2 h after treatment with 0.5 or 3 Gy or without treatment. (B) p53 stabilization was investigated 1 and 2 h after exposure to 0.5 Gy. Non-irradiated cells were harvested 1 h after mock-irradiation. (C) Phosphorylation of Chk2 assessed by the mobility shift of the protein band. Cells were collected 2 h after treatment with 3 or 10 Gy or without treatment.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
ATLD is a very rare disease, with only six cases described previously: four in the UK and two in Italy (20Go,27Go,28Go). The neurological presentation of these patients is very similar to that of AT patients. Two distinct genes, encoding for proteins involved in a same pathway, are responsible for the two disorders: ATM for AT and MRE11 for ATLD (20Go,33Go). Most disease-causing mutations in AT patients correspond to truncating mutations in the ATM gene, resulting in an unstable ATM protein. Very few cases of AT patients carrying two missense sequence alterations have been reported, expressing a stable, albeit, mutant ATM protein and showing a milder or classical AT clinical phenotype (34Go,35Go). Among the six ATLD patients described, two carry a homozygous mutation in the MRE11 gene (ATLD1 and ATLD2), leading to the expression of a C-terminally truncated Mre11 protein, missing the last 76 amino acids. The four other patients are compound heterozygotes, expressing a full-length mutant Mre11 protein and showing a milder phenotype (20Go,27Go–29Go).

The new ATLD patients identified here belong to three distinct Saudi Arabian families and carry a homozygous mutation in the MRE11 gene (630G->C, W210C). There is no known connection between the families. However, they reside in the central region of Saudi Arabia, which is surrounded by an ocean of sand and has been geographically isolated for many centuries. The patients were initially ascertained from the combination of early-onset, slowly progressive, ataxia plus ocular apraxia. They represent the largest set of ATLD patients identified to date and allow confirming and refining the features of this novel clinical entity. Age at onset is almost similar to that of AT, but disease progression is slower. Disease progression at later ages may include subsequent involvement of the basal ganglia. Most notably, certain extraneurological features are missing, such as telangiectasia, raised AFP level and reduced immunoglobulin levels. All patients had dysarthria. The clinical observations on this set of patients, together with those from previously reported cases, establish that ATLD is not associated with recurrent pulmonary infections due to immune deficiency, nor to susceptibility to lymphoid or other tumors or to skin abnormalities, which is of importance for prognosis formulation. Our patients present with some differences with the previously reported ATLD patients, namely, the absence of translocations on karyotyping two patients and the presence of microcephaly in four out of eight investigated patients, the latter feature being reminiscent of the clinical presentation of NBS. Whether these differences are specific to the W210C mutation or only reflect the wide spectrum of the disease needs further investigation. The cellular consequences of the amino acid change at position 210, located in the nuclease domain of the Mre11 protein, in terms of Mre11, Nbs1 and Rad50 protein level, and cellular response to ionizing radiation have been determined in fibroblast cultures established from two individuals.

These cell cultures both showed high levels of Mre11 and Rad50 proteins, compared with cells from normal individuals but a very low level of the Nbs1 protein. The genotyping data of the whole genome scan for family 1 excludes linkage of the disease with the NBS1 gene, because none of the patients is homozygous for NBS1 flanking markers and patient 1 inherited a different paternal haplotype, comprising the entire NBS1 region, than her affected siblings (data not shown). In order to rule out the possibility that in the two individuals studied NBS1 mutations might be the underlying cause of this cellular phenotype, the NBS1 cDNA from each culture was screened by direct sequencing and no mutations were detected. The ATLD7 fibroblasts carried four known NBS1 polymorphisms (RS1061302, RS709816, RS1805794 and RS1063045) that were not detected in ATLD8.

The ATLD cells described to date all express variable Mre11 protein levels, depending on their MRE11 genotype, and reduced levels of Nbs1 and Rad50. The presence of a truncating mutation in the MRE11 gene was associated with lower levels of the MRN proteins, compared with missense mutations (20Go,27Go,29Go). The homozygous G->C mutation at nucleotide 630 in ATLD7 and ATLD8 cells does not appear to affect the level of Mre11 protein expression but results in a destabilization of the MRN complex. One possible explanation for this observation is that the stable Mre11 mutant protein is able to interact with Rad50 but not with the Nbs1 protein. Preliminary results in support of this possibility have been obtained from immunoprecipitation experiments. Rad50 could be immunoprecipitated by an antibody against Mre11 from protein extracts prepared from both the new ATLD cell lines and a normal cell line. However, only in the protein extracts from the normal line could an interaction between NBS1 and Mre11 be detected using such an approach (data not shown). The Nbs1-interaction domain on Mre11 is not precisely identified but was found to be localized in the N-terminal 319 amino acids (20Go,36Go). Stewart et al. (20Go) showed that the Mre11 amino acid substitution of Asp to Ser at position 117 in cells from ATLD3 and 4 patients affects Nbs1 but not Rad50 binding to Mre11 protein. These results were confirmed in vitro by Lee et al. (10Go). The amino acid substitution of Trp to Cys at position 210 in the new ATLD cell lines, which also falls within this region, could selectively disturb the Mre11–Nbs1 interaction, leading to the instability of the MRN complex and lower level of Nbs1 protein. The underlying causes of the comparative over-expression of Mre11 and Rad50 remain to be determined but might be because of an altered half-life of these proteins or to post-translational phenomenon such as modification of the protein degradation pathways.

In order to determine whether the MRN complex was functional in ATLD7 and ATLD8 cells, the cellular response to DNA damage was investigated after exposure to IR. As observed in the ATLD cells already described and in NBS cells (37Go), no Mre11 or Nbs1 foci were formed in cells from the new ATLD patients 8 h after exposure to IR. At present, the physiological role of damage-induced foci is not clear, but these late MRN foci have been shown to reflect the presence of slowly repaired or irreparable DNA DSBs (22Go,32Go). Consistent with the loss of function of the MRN complex, as measured by the absence of IR-induced foci, cells from the new ATLD patients showed a cellular radiosensitivity intermediate between that of cells from normal individuals and that of AT and NBS cells, after exposure at 2 and 4 Gy. Among the ATLD patients examined, there appears to be an association between the cell survival and the type of mutation present. The ATLD1 and 2 cell lines, which carry two truncating MRE11 mutations, had the lowest cell survival, whereas the cell lines carrying a combination of one missense and one truncating mutation (ATLD3 and 4) or two missense mutations (ATLD7 and ATLD8) showed a more moderate radiosensitivity (20Go,27Go).

Recent work has suggested that the MRN complex may be required for full ATM activation, as demonstrated by defective ATM activation and defective phosphorylation of ATM targets such as p53-ser15, Chk2-thr68, Smc1-ser966 and 53BP1 in ATLD cells after exposure to DNA DSB-inducing agents (7Go–9Go,11Go,13Go,38Go) or Mre11 degradation by viral infection (7Go). ATLD7 and ATLD8 cells showed an ATM activation defect in a dose-dependent manner, as demonstrated by the formation of ATM serine 1981 phosphorylated foci, which was slightly reduced after exposure to 3 Gy but abrogated after exposure to 0.5 Gy. This observation correlated with a reduced p53 stabilization observed only after exposure to 0.5 Gy and a defect in p53-ser15 phosphorylation following a 0.5 or 3 Gy irradiation. Interestingly, phosphorylation of Chk2 was also abrogated in these cells, even after irradiation doses up to 10 Gy. This observation may be explained by the previously described Nbs1 dependence of this phosphorylation in cells expressing a low level of Nbs1 protein (8Go,13Go,39Go).

The dose dependence observed in the DNA damage response could be explained by the redundancy of several signaling pathways, with, in the case of ATM-dependent pathways, a probable intervention of other kinases such as ATR or DNA-PK which may mask the effect of an ATM loss, particularly after high-treatment doses (13Go,40Go). Phosphorylation of H2AX histone by ATM and DNA-PK is one of the first events occurring, following the formation of a DSB by IR (14Go,40Go,41Go). It has been shown that {gamma}H2AX and MRN foci co-localize and that H2AX is required for MRN foci formation (42Go) but the ability of ATLD cells to form {gamma}H2AX foci has not been previously reported. We showed here that 30 min after a 0.5 or 3 Gy irradiation, {gamma}H2AX foci are formed in ATLD7, ATLD8 and ATLD2 cells as in normal cells. This observation is in agreement with the fact that the presence of a functional MRN complex is not required for the formation of {gamma}H2AX foci, as already suggested by the ability of NBS cells to form these foci (8Go,19Go and this work).

The reduced ATM activation in ATLD cells, as measured by phosphorylation of ATM-ser 1981, with consequently a defective phosphorylation of the ATM kinase targets p53 and Chk2, tends to support the hypothesis of the requirement of a functional MRN complex for an optimal activation of the ATM kinase activity, which acts both upstream and downstream of ATM in DNA DSB signaling pathways (43Go,44Go and reviewed in 29Go). The relative contribution of individual components of the complex is difficult to assess but our results add support to recent findings showing that the modulation of the timing and the amplitude of ATM activation by Nbs1 in response to low doses of IR requires the ability of Nbs1 to interact with Mre11 (9Go,11Go) and the nuclease activity of Mre11 (13Go).

It has been shown that NBS and ATLD cells present a defect in intra-S phase checkpoints although less severe than for AT cells, but the presence of defects in G1 and G2 checkpoints has not been investigated in human ATLD cells. The role of the Nbs1 protein in G1 and G2 checkpoints control remains controversial and would appear to be dose dependent (reviewed in 23Go). Phosphorylations of p53 after irradiation lead to its activation and stabilization and thus to the control of cell cycle arrest in G1 (2Go,4Go). Examining the cell distribution in the G0/G1 and G2/M cell cycle compartments after exposure to 3 Gy, we found that while ATLD7 cells showed a cell cycle progression similar to that of normal cells, ATLD2 and ATLD8 cells presented a slightly defective G1 arrest. This observation correlates with the defect we observed in p53 phosphorylation and stabilization in these two cell cultures after exposure to a low-dose irradiation, which was less severe in the ATLD7 than in the ATLD8 and ATLD2 cells.

In conclusion, we have identified 10 new ATLD patients from unrelated families carrying a homozygous W210C missense mutation in the MRE11 gene, leading to the expression of an MRN protein complex with functional abnormalities. Our results demonstrate that this novel mutation results again in a hypomorphic protein, consistent with the mouse model data that indicate that a null mutation would be embryonic lethal. Although the two studied patients carry the same MRE11 mutation, qualitative differences were noted between the two corresponding cell cultures in terms of their cellular response to IR, suggesting that additional factors in the genetic background may impact on this response.

Alterations in the cellular response to ionizing radiation, demonstrated by defective ATM kinase activation and consequently reduced ATM target phosphorylation, radiosensitivity and cell cycle arrest defect were observed with a noticeable influence on these different end points, depending on the irradiation dose used. The cellular phenotype described here is very similar to that described for ATLD patients with heterozygous compound mutations, despite the presence of high levels of Mre11 and Rad50 proteins. This study emphasizes the importance of functional interactions among the three proteins of the MRN complex and lends further support to a role for this complex as a sensor of DNA DSBs, acting upstream of ATM. It also underlines the importance of using low-treatment doses to study the relationships between various proteins acting in the response to DNA damage.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Subjects
We studied three Saudi Arabian families with patients presenting early-onset ataxia plus oculomotor apraxia. In all three families, the parents are first-degree cousins. The clinical features of each patient are summarized in Table 1. No patient examined had telangiectasia, recurrent pulmonary infections, lymphoid or other tumors. Patients from all three families had normal levels of serum AFP, carcinoembryonic antigens and immunoglobulins. All patients had normal levels of serum albumin and cholesterol, including the patients of family 1 who have had clinical symptoms of the disease for >15 years. In family 1, four individuals, including two monozygotic male twins (patients 3 and 4), presented with progressive cerebellar ataxia with sitting or walking delay and ocular apraxia. With age, they developed facial dyskinesia (abnormal and involuntary facial movements similar to those seen in tardive dyskinesia), and masked face, indicating basal ganglia involvement and now have reduced tendon reflexes. Two other children died at 40 days and 1 year of age, respectively, from unknown causes. DNA samples of the patients, the parents and the four of five healthy siblings were available for study. In family 2, gait and limb ataxia was noted in three children from 15 months of age and brisk tendon reflexes from 2 years of age. The disease has progressed and they now have ocular apraxia and microcephaly. DNA samples of the patients, the parents and the three healthy siblings were available for study. In family 3, three children presented with ataxia, ocular apraxia and failure to thrive, with two patients having reduced tendon reflexes. The youngest patient also has microcephaly.

Genotyping and linkage analysis
Blood samples were obtained with informed consent. Genomic DNA was extracted from peripheral blood leukocytes using Qiagen DNA isolation kits. A whole-genome screen was initiated with family 1 at the Centre National de Génotypage (Evry, France) using a microsatellite marker set developed and commercialized by Applied Biosystems. (ABI linkage mapping set version 2, medium density set 10). This set comprises 400 fluorescently labeled microsatellite markers selected from the Généthon human linkage map (45Go), with an average spacing of 10 cM and an average heterozygosity of 75%. PCR-multiplex protocol and fragment analysis were performed as described (46Go). Additional CA/TG microsatellite markers from the Généthon human linkage map (45Go) were amplified with a universal fluoresceinated primer as described (46Go). PCR products were resolved on ABI 377 or ABI 3100 DNA sequencers (Applied Biosystems) and analyzed using the ABI PRISM GeneScan analysis software.

Part of the linkage power of families 1 and 2 resides in the consanguinity loop and linkage is supported when the patients are homozygous for a rare haplotype (30Go). This information was included in a two-point LOD score calculation by considering the non-recombinant haplotype as a single locus (30Go). Two-point LOD scores with consanguinity loops were calculated by using the MLINK program of the FASTLINK package (31Go). We assumed a fully penetrant autosomal recessive mode of inheritance, and a gene frequency of 0.001 that certainly represents an upper limit for this rare condition.

Sequence analysis
All 20 exons and flanking intronic sequences of MRE11 were PCR amplified from the DNA of patients from families 1 and 2 (primers and PCR conditions available on request). PCR products were purified with the NucleoSpin® (Extract 2 in 1 kit (Macherey-Nagel GmbH and Company). Sequencing was done using the ABI Prism Big DyeTM Terminator version 2.0 ready reaction cycle sequencing kit (Applied Biosystems). Reaction products were run on an automated DNA sequencer (model 3100 or 3700, Applied Biosystems). The presence or absence of the missense mutation was tested by DHPLC analysis of 200 control DNA samples from European and Arabian populations.

Total RNA extracted from the primary fibroblast cultures of ATLD7 and ATLD8, after treatment of the cell cultures with puromycin (100 µg/ml for a 6 h treatment period) (Sigma) using the Trizol reagent (Invitrogen Corporation) was reverse transcribed using the iScript cDNA synthesis kit, according to the manufacturer's instructions (Bio-Rad). The reaction product was used as a template to amplify the NBS1 cDNA in three overlapping fragments, which were subsequently sequenced as described. Primers and PCR conditions are available on request.

Cell lines and treatment
Primary fibroblast cultures were used throughout. Primary fibroblasts were established from a skin biopsy of patients 3 and 7 (lines ATLD7 and ATLD8, respectively). Skin biopsies were obtained with informed consent.

CZD82H is a cell culture established from an NBS patient of polish origin, AT1BR and 93RD469 are AT cell cultures and 90RD364, 85RD469, MRC5, 1BR3 and 251BR cells are control primary fibroblasts. The ATLD2 cell culture carries a homozygous truncating mutation in the MRE11 gene (20Go).

Fibroblasts were routinely maintained at 37°C, in a humidified incubator with 5% CO2, in exponential growth by dilution twice a week in minimum essential medium (Gibco, Invitrogen Corporation) or Dulbecco modified minimum essential medium (93RD346, CZD82H and ATLD2) supplemented with 10% fetal calf serum (Integro b.v,) and penicillin and streptomycin (100 µg/ml, Gibco, Invitrogen Corporation).

Exposures to ionizing radiation were carried out with a 137Cs source, at a dose rate of about 5 Gy/min.

Cell survival
The radiation survival of the fibroblasts was measured by their relative growth 12 days post-irradiation. The cells were seeded at 1x105 cells/flask the day before irradiation and cell survival was assessed after exposure to 0, 1, 2 and 4 Gy by counting the number of living cells using trypan blue exclusion. The number of viable cells in the non-irradiated culture 12 days post-irradiation was considered as 100% cell survival. Experiments were performed at least twice on each cell line with two flasks for each irradiation dose in each experiment.

Western blot analysis
All western blots analyses were carried out with total protein extracts prepared from control or irradiated fibroblasts. Briefly, cells were collected by trypsinization, washed twice in PBS and lysed in low-salt buffer (10 mM Hepes/KOH pH 7.5, 10 mM NaCl, 25 mM KCl, 1.1 mM MgCl2, 0.1 mM EDTA pH 7.5, 0.2 mg/ml pefabloc® (Boehringer, Roche Diagnostics), 2 µg/ml leupeptin, 1 mM DTT) for 5 min on ice. After two 5 min incubations in dry ice/methanol and a 37°C water bath successively, the supernatant was collected after centrifugation for 10 min at 10 621 g (4°C) and its protein concentration determined using the Bio-Rad protein assay. A 20–40 µg of this protein extract was separated on a 10% biphasic SDS–PAGE and transferred overnight (30 V) onto a PVDF membrane (Roche Diagnostics) using a Bio-Rad Trans-Blot Cell.

After 2 h incubation with the primary antibodies, the membranes were probed with peroxidase-conjugated goat anti-rabbit or anti-mouse IgG (DAKO) and developed using a chemiluminescence procedure (Amersham).

The p53-ser15 phosphorylation was examined using a specific p53-phosphoserine 15 antibody (#9284, Cell Signaling Technology, 1/1000 dilution), and the Chk2 phosphorylation was detected by the mobility shift of the protein using a specific Chk2 antibody (ab477, Abcam, 1/1500 dilution). The expression levels of p53 (DO7, Dako, 1/3000 dilution), Rad50 (ab89, Abcam, 1/500 dilution), Mre11 (ab397, Abcam, 1/10 000 dilution) and Nbs1 (ab398, Abcam, 1/10 000 dilution) proteins were determined by densitometry, after correction for protein loading using Ku80 (AHP317, Serotec, 1/40 000 dilution) or actin (#69100, ICN Biomedicals, 1/15 000 dilution) as an internal standard. Measurements were made at least twice from a minimum of two independently prepared protein extracts.

Immunofluorescence
Cells were seeded onto eight-well lab-tek II chamber slides (#154534, Polylabo) and treated with gamma irradiation at 0.5, 3 or 10 Gy or mock-irradiated 24 h later. After various recovery times, the cells were processed for immunofluorescence staining. Before fixation, the in situ cell fractionation procedure was performed as previously described (32Go). Briefly, the cells were washed twice with PBS (w/o Mg, Ca), incubated in cytoskeleton buffer (10 mM PIPES pH 6.8, 100 mM NaCl, 300 mM sucrose, 3 mM MgCl2, 1 mM EGTA, 0.5% Triton X-100) for 5 min on ice, followed by incubation in cytoskeleton stripping buffer (10 mM Tris–HCl pH 7.4, 10 mM NaCl, 3 mM MgCl2, 1% Tween 40, 0.5% sodium deoxycholate) for 5 min on ice. After two washes with PBS, the cells were fixed in Streck tissue fixative (Streck Laboratories) for 30 min at room temperature and permeabilized in 0.5% Triton X-100 solution for 5 min on ice. Cells were then blocked with 5% BSA in PBS for 30 min and incubated with primary antibody (1/200 dilution in 5% BSA/PBS) for 1 h at 37°C or overnight at 4°C, washed twice with PBS and incubated 45 min at 37°C in secondary antibody [fluorescein or rhodamine-conjugated affinipure F(ab')2 fragment anti-rabbit IgG (H+L), Jackson ImmunoResearch, 1/200 dilution]. Cells were then washed, counterstained with 4',6'-diamidino-2-phenylindole (Sigma, 0.05 µg/ml) for 5 min and the lab-tek was mounted with Vectashield (Vector).

Primary antibodies were directed against phospho-H2AX (#07-164, Upstate), ATM phospho S1981 (ab 2888, Abcam), Mre11 (ab397, Abcam) and Nbs1 (ab 398, Abcam).

Images were captured by using an Axioplan2 fluorescence microscope (Zeiss) equipped with a 50 W mercury lamp and Isis software (Metasystems Gmbh).

Cell cycle analysis
Cells mock-treated or treated with 3 Gy of ionizing radiation were collected by trypsinization 0, 14 and 24 h post-irradiation. After washing with PBS, cells were treated with propidium iodide using the CycleTestTM Plus Kit (Becton Dickinson), according to the manufacturer's instructions. The DNA content was analyzed by flow cytometry (FACSCalibur, Becton Dickinson), using the CellQuest software; 10 000 events were analyzed for each sample. The percentage of cells in each phase of the cell cycle was determined using the ModFit program. Experiments were performed at least in duplicate on each cell line.

Statistical methods
The differences in p53 and MRN proteins expression, relative cell survival and percentages of cells in cell cycle phases, between the fibroblast cultures established from the normal donors, the AT, ATLD and NBS patients have been tested by the analysis of the variance (ANOVA). For the inter-group comparison, significant heterogeneity in the variance was assessed by the Bartlett test, and when necessary, transformations of the variables were applied: square root of the proportion for Nbs1 basal level and cell survival after exposure to 4 Gy. For comparison of cell percentages in cell cycle phases, it was impossible to stabilize the variance, so a Mann–Whitney U-test was used. All the statistical computations were performed using the STATISTICA software (version 5.97, Statsoft).


   ACKNOWLEDGEMENTS
 
We wish to thank L. Reutenauer, E. Troesch, F. Ruffenach, I. Colas, S. Vicaire, B. Chapot and N. Moullan for excellent technical help, Dr C. Bétard and the Centre National de Génotypage for whole genome genotyping, Dr Ghassan Zidan, Cytogenetics Laboratory, King Khalid Iniversity Hospital, Riyadh for help with handling skin biopsies and Dr C. Arlett, Dr K. Chrzanowska, Dr W.J. Keijer and Dr A.M.R. Taylor for cell lines. Genetic studies were supported by funds from the Institut National de la Santé et de la Recherche Médicale, the Centre National de la Recherche Scientifique, the Hôpitaux Universitaires de Strasbourg (PHRC régional), and the GIS-Maladies Rares (SPATAX Research Network Grant 4MR12FA004DS). M.G. was supported by a fellowship from the Association Française de l'Ataxie de Friedreich. Part of this work was carried out under the tenure of an IARC special training award to M.F.


   FOOTNOTES
 
{dagger} The authors with it to be known that, in their opinion, the first two authors should be regarded as joint First Authors. Back

{ddagger} Present address: Laboratoire ‘Protéines du Cytosquelette’, Institut de Biologie Structurale, 41, rue Jules Horowitz, 38027 Grenoble Cedex 1, France. Back


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

  1. Bakkenist, C.J. and Kastan, M.B. (2003) DNA damage activates ATM through intermolecular autophosphorylation and dimer dissociation. Nature, 421, 499–506.[CrossRef][Medline]

  2. Canman, C.E., Lim, D.S., Cimprich, K.A., Taya, Y., Tamai, K., Sakaguchi, K., Appella, E., Kastan, M.B. and Siliciano, J.D. (1998) Activation of the ATM kinase by ionizing radiation and phosphorylation of p53. Science, 281, 1677–1679.[Abstract/Free Full Text]

  3. van den, B.M., Bree, R.T. and Lowndes, N.F. (2003) The MRN complex: coordinating and mediating the response to broken chromosomes. EMBO Rep., 4, 844–849.[CrossRef][ISI][Medline]

  4. Banin, S., Moyal, L., Shieh, S., Taya, Y., Anderson, C.W., Chessa, L., Smorodinsky, N.I., Prives, C., Reiss, Y., Shiloh, Y. and Ziv, Y. (1998) Enhanced phosphorylation of p53 by ATM in response to DNA damage. Science, 281, 1674–1677.[Abstract/Free Full Text]

  5. Matsuoka, S., Rotman, G., Ogawa, A., Shiloh, Y., Tamai, K. and Elledge, S.J. (2000) Ataxia telangiectasia-mutated phosphorylates Chk2 in vivo and in vitro. Proc. Natl Acad. Sci. USA, 97, 10389–10394.[Abstract/Free Full Text]

  6. Nakagawa, K., Taya, Y., Tamai, K. and Yamaizumi, M. (1999) Requirement of ATM in phosphorylation of the human p53 protein at serine 15 following DNA double-strand breaks. Mol. Cell. Biol., 19, 2828–2834.[Abstract/Free Full Text]

  7. Carson, C.T., Schwartz, R.A., Stracker, T.H., Lilley, C.E., Lee, D.V. and Weitzman, M.D. (2003) The Mre11 complex is required for ATM activation and the G2/M checkpoint. EMBO J., 22, 6610–6620.[CrossRef][ISI][Medline]

  8. Girard, P.M., Riballo, E., Begg, A.C., Waugh, A. and Jeggo, P.A. (2002) Nbs1 promotes ATM dependent phosphorylation events including those required for G1/S arrest. Oncogene, 21, 4191–4199.[CrossRef][ISI][Medline]

  9. Horejsi, Z., Falck, J., Bakkenist, C.J., Kastan, M.B., Lukas, J. and Bartek, J. (2004) Distinct functional domains of Nbs1 modulate the timing and magnitude of ATM activation after low doses of ionizing radiation. Oncogene, 23, 3122–3127.[CrossRef][ISI][Medline]

  10. Lee, J.H., Ghirlando, R., Bhaskara, V., Hoffmeyer, M.R., Gu, J. and Paull, T.T. (2003) Regulation of Mre11/Rad50 by Nbs1: effects on nucleotide-dependent DNA binding and association with ataxia-telangiectasia-like disorder mutant complexes. J. Biol. Chem., 278, 45171–45181.[Abstract/Free Full Text]

  11. Lee, J.H., Xu, B., Lee, C.H., Ahn, J.Y., Song, M.S., Lee, H., Canman, C.E., Lee, J.S., Kastan, M.B. and Lim, D.S. (2003) Distinct functions of Nijmegen breakage syndrome in ataxia telangiectasia mutated-dependent responses to DNA damage. Mol. Cancer Res., 1, 674–681.[Abstract/Free Full Text]

  12. Mochan, T.A., Venere, M., DiTullio, R.A., Jr and Halazonetis, T.D. (2003) 53BP1 and NFBD1/MDC1-Nbs1 function in parallel interacting pathways activating ataxia-telangiectasia mutated (ATM) in response to DNA damage. Cancer Res., 63, 8586–8591.[Abstract/Free Full Text]

  13. Uziel, T., Lerenthal, Y., Moyal, L., Andegeko, Y., Mittelman, L. and Shiloh, Y. (2003) Requirement of the MRN complex for ATM activation by DNA damage. EMBO J., 22, 5612–5621.[CrossRef][ISI][Medline]

  14. Burma, S., Chen, B.P., Murphy, M., Kurimasa, A. and Chen, D.J. (2001) ATM phosphorylates histone H2AX in response to DNA double-strand breaks. J. Biol. Chem., 276, 42462–42467.[Abstract/Free Full Text]

  15. Rogakou, E.P., Pilch, D.R., Orr, A.H., Ivanova, V.S. and Bonner, W.M. (1998) DNA double-stranded breaks induce histone H2AX phosphorylation on serine 139. J. Biol. Chem., 273, 5858–5868.[Abstract/Free Full Text]

  16. Fernandez-Capetillo, O., Lee, A., Nussenzweig, M.C. and Nussenzweig, A. (2004) H2AX: the histone guardian of the genome. DNA Repair, 3, 959–967.[CrossRef][Medline]

  17. Fernandez-Capetillo, O., Chen, H.T., Celeste, A., Ward, I., Romanienko, P.J., Morales, J.C., Naka, K., Xia, Z., Camerini-Otero, R.D., Motoyama, N. et al. (2002) DNA damage-induced G2-M checkpoint activation by histone H2AX and 53BP1. Nat. Cell Biol., 4, 993–997.[CrossRef][ISI][Medline]

  18. Fernandez-Capetillo, O., Celeste, A. and Nussenzweig, A. (2003) Focusing on foci: H2AX and the recruitment of DNA-damage response factors. Cell Cycle, 2, 426–427.[Medline]

  19. Kobayashi, J., Tauchi, H., Sakamoto, S., Nakamura, A., Morishima, K., Matsuura, S., Kobayashi, T., Tamai, K., Tanimoto, K. and Komatsu, K. (2002) NBS1 localizes to gamma-H2AX foci through interaction with the FHA/BRCT domain. Curr. Biol., 12, 1846–1851.[CrossRef][ISI][Medline]

  20. Stewart, G.S., Maser, R.S., Stankovic, T., Bressan, D.A., Kaplan, M.I., Jaspers, N.G., Raams, A., Byrd, P.J., Petrini, J.H. and Taylor, A.M. (1999) The DNA double-strand break repair gene hMRE11 is mutated in individuals with an ataxia-telangiectasia-like disorder. Cell, 99, 577–587.[CrossRef][ISI][Medline]

  21. Ward, I.M., Minn, K., Jorda, K.G. and Chen, J. (2003) Accumulation of checkpoint protein 53BP1 at DNA breaks involves its binding to phosphorylated histone H2AX. J. Biol. Chem., 278, 19579–19582.[Abstract/Free Full Text]

  22. Maser, R.S., Monsen, K.J., Nelms, B.E. and Petrini, J.H. (1997) hMre11 and hRad50 nuclear foci are induced during the normal cellular response to DNA double-strand breaks. Mol. Cell. Biol., 17, 6087–6096.[Abstract]

  23. Tauchi, H., Matsuura, S., Kobayashi, J., Sakamoto, S. and Komatsu, K. (2002) Nijmegen breakage syndrome gene, NBS1 and molecular links to factors for genome stability. Oncogene, 21, 8967–8980.[CrossRef][ISI][Medline]

  24. Rotman, G. and Shiloh, Y. (1998) ATM: from gene to function. Hum. Mol. Genet., 7, 1555–1563.[Abstract/Free Full Text]

  25. Shiloh, Y. (2003) ATM and related protein kinases: safeguarding genome integrity. Nat. Rev. Cancer, 3, 155–168.[CrossRef][ISI][Medline]

  26. Digweed, M., Reis, A. and Sperling, K. (1999) Nijmegen breakage syndrome: consequences of defective DNA double strand break repair. Bioessays, 21, 649–656.[CrossRef][ISI][Medline]

  27. Delia, D., Piane, M., Buscemi, G., Savio, C., Palmeri, S., Lulli, P., Carlessi, L., Fontanella, E. and Chessa, L. (2004) MRE11 mutations and impaired ATM-dependent responses in an Italian family with Ataxia-telangiectasia like disorder (ATLD). Hum. Mol. Genet., 13, 2155–263.[Abstract/Free Full Text]

  28. Pitts, S.A., Kullar, H.S., Stankovic, T., Stewart, G.S., Last, J.I., Bedenham, T., Armstrong, S.J., Piane, M., Chessa, L., Taylor, A.M. and Byrd, P.J. (2001) hMRE11: genomic structure and a null mutation identified in a transcript protected from nonsense-mediated mRNA decay. Hum. Mol. Genet., 10, 1155–1162.[Abstract/Free Full Text]

  29. Taylor, A.M., Groom, A. and Byrd, P.J. (2004) Ataxia-telangiectasia disorder (ATLD)-its clinical presentation and molecular basis. DNA Repair, 3, 1219–1225.[Medline]

  30. Ben Hamida, C., Doerflinger, N., Belal, S., Linder, C., Reutenauer, L., Dib, C., Gyapay, G., Vignal, A., Le Paslier, D., Cohen, D. et al. (1993) Localization of Friedreich ataxia phenotype with selective vitamin E deficiency to chromosome 8q by homozygosity mapping. Nat. Genet., 5, 195–200.[CrossRef][ISI][Medline]

  31. Cottingham, R.W. Jr., Idury, R.M. and Schaffer, A.A. (1993) Faster sequential genetic linkage computations. Am. J. Hum. Genet., 53, 252–263.[ISI][Medline]

  32. Mirzoeva, O.K. and Petrini, J.H. (2001) DNA damage-dependent nuclear dynamics of the Mre11 complex. Mol. Cell. Biol., 21, 281–288.[Abstract/Free Full Text]

  33. Savitsky, K., Bar-Shira, A., Gilad, S., Rotman, G., Ziv, Y., Vanagaite, L., Tagle, D.A., Smith, S., Uziel, T., Sfez, S. et al. (1995) A single ataxia telangiectasia gene with a product similar to PI-3 kinase. Science, 268, 1749–1753.[Abstract/Free Full Text]

  34. Angele, S., Lauge, A., Fernet, M., Moullan, N., Beauvais, P., Couturier, J., Stoppa-Lyonnet, D. and Hall, J. (2003) Phenotypic cellular characterization of an ataxia telangiectasia patient carrying a causal homozygous missense mutation. Hum. Mutat., 21, 169–170.[Medline]

  35. Lavin, M.F., Scott, S., Gueven, N., Kozlov, S., Peng, C. and Chen, P. (2004) Functional consequences of sequence alterations in the ATM gene. DNA Repair, 3, 1197–1205.[Medline]

  36. Desai-Mehta, A., Cerosaletti, K.M. and Concannon, P. (2001) Distinct functional domains of nibrin mediate Mre11 binding, focus formation and nuclear localization. Mol. Cell. Biol., 21, 2184–2191.[Abstract/Free Full Text]

  37. Carney, J.P., Maser, R.S., Olivares, H., Davis, E.M., Le Beau, M., Yates, J.R. III, Hays, L., Morgan, W.F. and Petrini, J.H. (1998) The hMre11/hRad50 protein complex and Nijmegen breakage syndrome: linkage of double-strand break repair to the cellular DNA damage response. Cell, 93, 477–486.[CrossRef][ISI][Medline]

  38. Lee, J.H. and Paull, T.T. (2004) Direct activation of the ATM protein kinase by the Mre11/Rad50/Nbs1 complex. Science, 304, 93–96.[Abstract/Free Full Text]

  39. Buscemi, G., Savio, C., Zannini, L., Micciche, F., Masnada, D., Nakanishi, M., Tauchi, H., Komatsu, K., Mizutani, S., Khanna, K. et al. (2001) Chk2 activation dependence on Nbs1 after DNA damage. Mol. Cell. Biol., 21, 5214–5222.[Abstract/Free Full Text]

  40. Stiff, T., O'Driscoll, M., Rief, N., Iwabuchi, K., Lobrich, M. and Jeggo, P.A. (2004) ATM and DNA-PK function redundantly to phosphorylate H2AX after exposure to ionizing radiation. Cancer Res., 64, 2390–2396.[Abstract/Free Full Text]

  41. Park, E.J., Chan, D.W., Park, J.H., Oettinger, M.A. and Kwon, J. (2003) DNA-PK is activated by nucleosomes and phosphorylates H2AX within the nucleosomes in an acetylation-dependent manner. Nucleic Acids Res., 31, 6819–6827.[Abstract/Free Full Text]

  42. Furuta, T., Takemura, H., Liao, Z.Y., Aune, G.J., Redon, C., Sedelnikova, O.A., Pilch, D.R., Rogakou, E.P., Celeste, A., Chen, H.T. et al. (2003) Phosphorylation of histone H2AX and activation of Mre11, Rad50 and Nbs1 in response to replication-dependent DNA d