MRE11 mutations and impaired ATM-dependent responses in an Italian family with ataxia-telangiectasia-like disorder

Revised Manuscript HMG-2004D-00154

MRE11 mutations and impaired ATM-dependent responses in an

Italian family with Ataxia-Telangiectasia Like Disorder (ATLD)

Domenico Delia1, Maria Piane2, Giacomo Buscemi1, Camilla Savio2, Silvia Palmeri 3, Patrizia Lulli2, Luigi

Carlessi1, Enrico Fontanella1, and Luciana Chessa2§

1 Department of Experimental Oncology, Istituto Nazionale Tumori, Via G. Venezian 1, 20133 Milano, Italy;

2Department of Experimental Medicine Pathology, II Faculty of Medicine, University “La Sapienza”, Roma,

Italy; 3Department of Neurological Sciences, Policlinico Le Scotte, University of Siena, 53100 Siena, Italy.

§Corresponding Author:

Prof. Luciana Chessa

II Faculty of Medicine, A.O. S. Andrea

Via di Grottarossa 1035

I-00189 Roma, Italy

Phone/fax: +39-06-80345258

[email protected]

Copyright © 2004 Oxford University Press

HMG Advance Access published July 21, 2004 by guest on February 7, 2016

http://hmg.oxfordjournals.org/

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ABSTRACT

Hypomorphic mutations of the MRE11 gene are the hallmark of the radiosensitive ataxia-

telangiectasia-like disorder (ATLD). Here we describe a new family with two affected siblings,

ATLD5 and ATLD6, now aged 37 and 36 respectively. They presented with late onset cerebellar

degeneration slowly progressing until puberty and absence of telangiectasias and were cancer-free.

Both patients were wild type for ATM and NBS1, but compound heterozygotes for MRE11 gene

mutations [1422C→A, T481K; 1714C→T, R571X]. The 1422C→A allele was inherited from the

mother, whereas the 1714C→T, paternally inherited, was apparently null as result of non-sense

mediated mRNA decay (NMD). Interestingly, the 1714C→T mutation is the same previously

identified in an unrelated English ATLD family (probands ATLD3 and ATLD4), suggesting an

important role for NMD in saving potentially lethal mutations. Lymphoblastoid cells (LCLs)

derived from ATLD5 and ATLD6 were normal for ATM, but defective for Mre11, Rad50 and Nbs1

(the MRN complex) protein expression. Their response to γ-radiation was abnormal, as evidenced

by the enhanced radiosensitivity, attenuated autophosphorylation of ATM-S1981 and

phosphorylation of the ATM targets p53-S15 and Smc1-S966, failure to form Mre11 nuclear foci,

and defective G1 checkpoint arrest. The fibroblasts but not LCLs from ATLD5 and ATLD6 showed

an impaired ATM-dependent Chk2 phosphorylation. These findings further underscore the

interconnection between ATM activity and MRN function, that rationalizes the clinical similarity

between A-T and ATLD.

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INTRODUCTION

Ataxia-Telangiectasia (A-T) is a pleiotropic autosomal recessive disorder characterized by

cerebellar ataxia, teleangiectasias, immunodeficiency, radiosensitivity and predisposition to

malignancy. Milder A-T cases, termed A-T Variants, present later onset of the disease and/or

moderate severity of clinical features and longer life span (1-3).

The common phenotypic features of classical A-T and A-T Variants are hypersensitivity to ionizing

radiation and radiomimetic drugs, defective cell cycle checkpoints and alterations of DNA double-

strand breaks (DBSs) repair, altogether contributing to genomic instability and cancer

predisposition. Classical A-T patients show homozygous or compound heterozygous mutations of

the ATM gene, that generally lead to the truncation of the protein product (4-9). ATM mutations

were also described in some A-T Variants and in one A-T Fresno patient (10).

More recently, four probands belonging to two unrelated English families with A-T variant

phenotype, defined as A-T like disorder (ATLD), have been found to carry mutations in the MRE11

gene (11). Of these, family 1 probands (ATLD1 and ATLD2) are affected by a severe form of the

disease and carry homozygous MRE11 mutations that lead to low levels expression of truncated

protein. Family 2 probands (ATLD3 and ATLD4) are affected by a milder form of the disease and

carry compound heterozygous MRE11 mutations that lead to the expression of a partially active

Mre11 protein (11).

The MRE11 gene encodes a protein with nuclease and intrinsic DNA binding activity that, through

interaction with Rad50 and Nbs1, forms the core of the MRN complex involved in DSB sensing,

DNA recombination and multiple cell cycle checkpoints (12, 13). The MRN complex proteins are

homogeneously diffuse in the nucleus of normal undamaged cells, but after treatment with

genotoxic agents are rapidly recruited at sites of DNA lesions, giving rise to the formation of

nuclear foci believed to play a role in DSBs processing and checkpoints signalling (13). In ATLD

cells, where the MRE11 mutations result in reduced expression of Mre11 as well as of Rad50 and

Nbs1 proteins, the formation of MRN foci is impaired. The recently generated Mre11ATLD1/ATLD1



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mice expressing the ATLD1 allele (1897 C→T, R633X) appear to recapitulate the phenotypic

features of ATLD, including radiation hypersensitivity, chromosomal instability and defective arrest

at G1-S, intra-S and G2-M checkpoints (14). Interestingly, these Mre11ATLD1/ATLD1 mice are not

prone to malignancy, indicating that the observed phenotypic defects are per se insufficient to

significantly enhance the initiation of tumorigenesis (14).

The response to DSBs primarily involves the activation of ATM kinase and phosphorylation of

several targets, eg: p53, Chk2, Brca1, Smc1, Nbs1, critical for cell cycle checkpoint activation,

DNA repair and apoptosis (15). Although the MRN complex is a target of ATM kinase, at least two

pieces of evidence have recently underscored the critical role of MRN in stimulating the catalytic

activity of ATM. Studies with ATLD have shown that Mre11 deficiency compromises the

radiation-induced autophosphorylation of ATM on serine 1981 as well as the phosphorylation of its

downstream targets, and this defect can be corrected by wild type MRE11 (16). In vitro biochemical

studies have provided additional mechanistic insights into the functional role and requirement of the

individual components of the MRN complex (eg: Nbs1, Mre11) in the stimulation of ATM activity

towards some substrates (17). This interdependent ATM/MRN relationship provides a mechanistic

explanation for the phenotypic similarity between A-T and ATLD (16).

Here we describe two siblings, ATLD5 and ATLD6, presenting with a slowly progressive

neurological syndrome initailly diagnosed as Ataxia without Telangiectasia [A(-T)], who have

germline mutations of the MRE11 gene. They were shown to be compound heterozygotes for

mutations 1422C→A, resulting in base exchange T481K, and 1714C→T, which generates a stop

signal at codon 571X. Interestingly, the paternally inherited mutant allele 1714C→T is subjected to

nonsense-mediated mRNA decay (NMD), as detected in an unrelated English ATLD family

(patients ATLD3 and ATLD4) with the same mutation (18). NMD is a cellular surveillance

mechanism that in most vertebrates selectively degrades mRNAs with premature termination

codons, reducing the amount of non-functional mRNA that would produce truncated proteins

potentially exerting dominant negative effects (19, 20). Cells from our patients show a number of



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phenotypic defects, including moderate chromosomal and clonogenic radiosensitivity, reduced

expression and function of the MRN complex, attenuated ATM kinase activation and

phoshorylation of some of its target, features these more similar to those observed in ATLD4 than

ATLD2 cells.



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RESULTS

Description of patients

The affected siblings (ATLD5, a 38-year-old man, and ATLD6, a 37-year-old woman) were born to

non-consanguineous parents. Their father died of stroke when 61-year-old, while their 59-year-old

mother is in good health. The maternal lineage included a grandmother that died of stroke, an aunt

affected by deaf-mutism and an uncle by mild oligophrenia and parkinsonism. The paternal lineage

included a grandmother and her two sisters who died from stroke and a grandfather who died from

bladder carcinoma.

ATLD5, the elder son, was normal until the age of 3 years, but then developed progressive

unsteadiness and by 6 years of age showed diffuse cerebellar signs, i.e. ataxic gait, delayed speech

and writing difficulties, choreoathetoid arm movements and oculomotor apraxia. The disease

progressed slowly till the age of 14 and then stabilized. The latest neurological examination at 36

years of age showed cerebellar dysarthria, oculomotor apraxia, ataxic gait with unaided walk for

few steps, choreoathethosis of the superior limbs, jerk nystagmus on horizontal and vertical gaze,

dysmetria, dyskinetic movements of mouth and slight dystonia of the hands, diffuse hypotonia,

reduced tendon reflexes in the arms, absent ankle jerks with flexor plantar responses.

ATLD6, the younger sister, had a normal psychomotor development, but by 6 years of age had

acquired unsteadiness and writing difficulties. The progression of the disease was similar to her

brother and resulted in dystonic movements of the face and hands along with cerebellar ataxia,

ocular apraxia and cerebellar dysarthria.

Laboratory findings, including immunoglobulins, alphafetoprotein, lysosomial enzymes,

lipoproteins and vitamin E levels, were in the normal range for both patients; EMG revealed a slight

motor sensory neuropathy. Repeated CT Scans performed in both patients at various times showed

cerebellar atrophy with a moderate size increase of the fourth ventricle, subsequently confirmed by

brain MRIs.



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Identification of MRE11 mutations

The search for ATM and NBS1 gene mutations failed to reveal any alterations. However, sequence

analysis of the cDNAs revealed an MRE11 missense mutation at codon 1442 (C→A; T481K) (Fig

1), also detected in the maternal cDNA. As the expression of the inherited paternal allele was

undetectable, the search for mutations was performed on the genomic DNA by sequencing each of

the MRE11 exons. This led to the identification of a single C→T base change in exon 15,

corresponding to nucleotide 1714 in the cDNA sequence, that introduces a premature stop codon

(R571X) predicted to encode a prematurely truncated protein of 65kDa. However, such product

was not detected in western blots of cell lysates from both patients, suggesting that the 1714 C→T

allele might undergo the nonsense-mediated mRNA decay. This appeared to be the case, since the

cDNA carrying the 1714 C→T mutation was detected only after prevention of NMD by treatment

of cells with anisomycin (Fig 1). When examined by Protein Truncation Test (PTT) this transcript

encoded the expected truncated Mre11 protein of 65kDa (data not shown). In summary, ATLD5

and ATLD6 are compound heterozygotes for MRE11 gene mutations [1422C→A, T481K;

1714C→T, R571X], and are null for the expression of the paternal allele because of NMD.

Radiosensitivity findings

Chromosomal breakage tests performed on peripheral blood lymphocytes from ATLD5 and ATLD6

showed an increased rate of radioinduced breaks (respectively 27 and 25 cells with breaks on 50

cells examined), intermediate between normal (16+2) and A-T (34+5) cells. No clonal

chromosomal abnormalities, like the t(7;14) translocation typical of A-T, were detected, and the rate

of spontaneous chromosome breakage was within the normal range. Colony survival assays

performed on the LCLs established from ATLD5 and ATLD6 showed an increased sensitivity to

1Gy of irradiation, relative to normal cells (Fig 2). Nevertheless, their radiosensitivity was less

pronounced compared with ATLD4 and ATLD2, the two previously reported cases with mild and

severe clinical features, respectively (11).



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Expression of the MRN complex proteins and ATM-dependent responses to radiation

Immunoblots were performed to determine the level of ATM, Mre11, Nbs1 and Rad50 proteins in

our ATLD cases also in comparison with the English cases. Both ATLD5 and ATLD6, like ATLD2

and ATLD4, expressed normal levels of ATM protein, a finding consistent with the failure to detect

ATM mutations in these cases. Conversely, the levels of Mre11 as well as those of Rad50 and Nbs1

were significantly reduced in ATLD5 and ATLD6, as in ATLD4 (Fig 3). ATLD2 appeared negative

for Mre11 and Rad50, whereas it showed residual levels of Nbs1 protein (Fig 3). Thus, the

underlying genetic defect in ATLD5 and ATLD6 compromises the expression of the MRN

complex components almost to the same extent as in ATLD4, but less severely than in ATLD2.

The MRN complex is recruited, along with other recognition and repair proteins, at sites of DSBs

giving rise to nuclear foci (21). As MRE11 mutations can impair the function and localization of

MRN complex (11), we determined the capacity of ATLD5 and ATLD6 to form nuclear foci.

Compared with normal cells, in which the constitutively diffuse Mre11 nuclear fluorescence

became localized in foci after irradiation, ATLD5 and ATLD6, besides showing a reduced Mre11

fluorescence, failed to form foci after irradiation, compatible with a defective MRN complex in

these cells (Fig 4).

Since the DNA damage-induced ATM activity, as monitored by the autophosphorylation of S1981,

is markedly attenuated in ATLD (16, 22), we determined occurrence of this defect in our ATLD

cases at 30min post-irradiation. Compared with normal cells, the ATM pS1981 signal detected in

ATLD5 and ATLD6 resulted reduced by 50% after 0.25Gy and 30-40% after 1Gy (Fig 5A), to

almost the same extent as in ATLD4. The ATM pS1981 signal was even more attenuated in

ATLD2 and undetectable in AT. Thus, the activation of ATM in our cases is only partially

impaired.

To determine the effect of this attenuated activity of ATM, we analysed the phosphorylation of its

downstream targets Chk2, p53 and Smc1. The initial phosphorylation of Chk2 on Thr68 by ATM

triggers additional phosphorylation steps that fully activate Chk2 (23). The phosphorylation of



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Chk2 is impaired in NBS cells with defects of the MRN complex due to inactivating mutations of

NBS1 gene (24). In response to IR doses >1Gy, ATLD5 and ATLD6 lymphoblastoid cells showed a

similar phosphorylation-related Chk2 mobility delay as normal cells, and this delay was also seen in

ATLD4, but not ATLD2 or AT cells (Fig 5B). Strikingly, however, the Chk2 mobility delay in

ATLD5 and ATLD6 appeared cell type specific as it was absent in the irradiated fibroblasts from

these patients (Fig 5C).

The phosphorylation of p53-Ser15 was analysed at 30 min post-IR, a time point when this event

mostly reflects the activity of ATM, rather than ATR (25, 26). To assess Ser15 phosphorylation

independently of changes in p53 protein levels, prior to irradiation the cells were treated with the

proteasome inhibitor LLnL to allow the accumulation of basal p53 (26). The results (Fig 6) showed

an 8-9 fold increase in p53-pS15 levels in normal cells, and only 1.2 fold in A-T cells, relative to

the untreated cell counterparts. In ATLD5 and ATLD6 cells, the phosho-Ser15 levels increased 5.2-

5.8 fold, as in NBS cells, whereas in ATLD4 and ATLD2 increased 3.1 and 2.3 fold, respectively.

ATM phosphorylates Smc1-S966, and this event is required for the radiation-induced S phase cell

cycle checkpoint arrest (27). Compared with untreated cells, the S966 phosphorylation signal in

ATLD5 and ATLD6 at 30 min after irradiation was lower than in LBC-N (1.9, 1.7 and 5.5 fold

increase, respectively), but this difference was not seen at 60 min (Fig 7A). However, ATLD2, and

to a lesser extent ATLD4, showed a defective S966 phosphorylation at both time points (Fig 7B).

Flow cytometric cell cycle analyses were performed to determine the outcome of the defective

ATM-p53 response on G1 checkpoint arrest. At 24hrs post irradiation, normal cells showed a

slightly reduced G1/G2-M ratio that was compatible with a normal G1 checkpoint, whereas A-T

cells showed a marked drop of G1 phase cells (thus causing an inversion of the G1/G2-M ratio)

indicative of a failure to arrest in G1 (Fig 8 and Table 2). Compatible with a defective G1 arrest,

ATLD5 and ATLD6 cells showed an inversion of the G1/G2-M ratio (0.86+0.15 and 0.77+0.11,

respectively). ATLD2, but not ATLD4 showed a similar defect (Fig 8).



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DISCUSSION

Inherited mutations of the MRE11 gene have been recently identified in four patients with ATLD,

an A-T Variant disorder presenting many of the clinical features of A-T, but often absence of

telangiectasias, moderate radiosensitivity, later onset and slow progression of the disease, longer

survival and no tumor recurrence (11). Among cases classified as A-T Variants, carriers of ATR

(28) and LIGASE IV (29) germline mutations have also been identified.

In this study we describe a new family in which heterozygous compound mutations of the MRE11

gene [1422C→A, T481K; 1714C→T, R571X] have been identified in the affected brother ND

sister, now aged 38 and 37, who were initially diagnosed as Ataxia without Telangiectasia patients.

Consistent with this diagnosis, these cancer-free patients did not exhibit ocular telangiectasias and

presented with mild and slowly progressing neurological dysfunctions that manifested when they

were 3 years old and stabilized when 14. Analysis of the samples from their parents showed that the

1422C→A allele, which causes a T→K aminoacid change at codon 481, was inherited from the

mother, whereas the 1714C→T allele, which results in a premature stop at codon 571, was

paternally inherited. Interestingly, the 1714C→T mutation could be only detected from the

sequencing of genomic DNA but not from cDNA, suggesting that this mutation destabilizes the

transcript by NMD. This was indeed the case, since the cDNA with the 1714C→T mutation was

recovered from cells treated with anysomicin, a drug that prevents mRNA decay. Worthnoting, the

1714C→T allele is the same identified in an unrelated English family (patients ATLD3 and

ATLD4) (11), and also in these cases the transcript is subjected to NMD (18). NMD is a

surveillance mechanism which eliminates the errors in the biogenesis of mRNA (20, 30, 31). The

decay of the transcripts containing premature termination codons prevents the expression of

potentially deleterious truncated proteins, mitigating the clinical severity (19). MRE11 is an

essential gene for survival and, although the six patients reported to date show variable degrees of

clinical severity as well as different amount of Mre11 protein, the milder cases, albeit unrelated,



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share the same 1714C→T null mutation, suggesting that NMD plays a role in saving potentially

lethal mutations.

The MRE11 mutations in our ATLD cases impaired not only the expression of Mre11, but also of

Rad50 and Nbs1 proteins, to the same extent as in ATLD4, but not as much as in ATLD2, where

these proteins were almost undetectable (11). Furthermore, these MRE11 mutations conferred

partial radiosensitivity, as demonstrated by colony survival assays, and disrupted the activity of the

MRN complex to form nuclear foci in response to radiation.

The MRN complex, besides being a target of ATM, is a direct inducer of ATM kinase. Accordingly,

in Mre11-deficient ATLD cells the autophosphorylation of ATM S1981 was found markedly

attenuated compared with normal cells, and this defect could be corrected by wild type MRE11

(16). Biochemical studies have further substantiated the role of MRN in ATM activation (17). The

attenuated activation of ATM in ATLD cells, resulting in a defective phosphorylation of ATM

targets, actually provides an explanation for the phenotypic similarities between A-T and ATLD

(16). The autophosphorylation of ATM-S1981 in irradiated ATLD5 and ATLD6 was attenuated,

though not as markedly as in A-T or ATLD2 cells. This attenuation was associated with reduced

phosphorylation of p53-S15, an event involved in p53 stabilization and transcriptional induction of

its target gene p21waf1, a key inhibitor of the G1-S checkpoint (25). This defective phosphorylation

of p53 correlated with failure of our ATLD cases to properly enforce a G1 arrest after irradiation.

Chk2, a kinase involved in multiple cell cycle checkpoints arrest after DNA damage (32), is

targeted by ATM on T68, and this event triggers the hyperphosphorylation and full activation of

Chk2. We have shown a normal hyperphosphorylation of Chk2 in LCLs from ATLD5 and ATLD6,

indicating that ATM, despite its reduced activity, is able to activate Chk2. Interestingly, however,

the hyperphosphorylation of Chk2 was impaired in the fibroblasts from these cases, suggesting that

the efficiency of ATM or access to its substrates may somehow exhibit a tissue specificity.

The phosphorylation of Smc1-S966 by ATM is required for the radiation-induced S phase cell cycle

checkpoint arrest (27). We have shown that the phosphorylation of this target of ATM is delayed in



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ATLD5 and ATLD6, suggesting that the attenuated activation of ATM has a limited effect on

Smc1.

Chromosomal instability is a common genetic defect of A-T, NBS and ATLD (33), yet neither

ATLD5 nor ATLD6 showed chromosomal translocations, in contrast to ATLD1/2 and ATLD3/4 in

which these abnormalities were seen in 8% and 1% of blood lymphocytes, respectively (11). This

result suggests that the genetic defect of the Italian cases is not so severe to impair chromosomal

stability. Although a major outcome of genome instability is cancer, the lack of tumors among the

English and Italian ATLD patients would suggest that Mre11 deficiency alone does not increase

cancer predisposition. This conclusion, clearly precluded by the limited human cohort number,

would be compatible with recent findings showing that Mre11ATLD1/ATLD1 mice exhibit

chromosomal instability but are not cancer prone (14).

In conclusion, we have identified two new ATLD siblings with heterozygous compound mutations

of the MRE11 gene causing defective expression and function of the MRN complex, impairement

of ATM activity and downstream signalling in response to ionizing radiation. These findings lend

further support to the interconnection between MRN and ATM.



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MATERIAL AND METHODS

Cell lines and treatments

Lymphoblastoid cell lines (LCLs) were established by EBV-immortalization of peripheral blood

lymphocytes from ATLD5 and ATLD6. Normal cells (LBC-N) were obtained from a normal donor;

A-T cells from patient AT52RM (34). The LCLs from ATLD2 and ATLD4 have been described

(11). The NBS-derived cell line GM07078 was purchased from Coriell Cell Repository (Camden,

N.J.). Skin biopsies from ATLD5 and ATLD6 were used to generate fibroblast cell lines. Cells were

cultured at 37°C in a CO2 incubator using RPMI 1640 supplemented with 15% of heat inactivated

fetal calf serum, 1% of glutamine and antibiotics.

Radiosensitivity analysis

Chromosome breakage analysis were performed on peripheral lymphocytes from patients and

normal age- and sex-matched controls. Cells were irradiated in the G2 phase with 0.25Gys

delivered at a rate of 70cGy/min with a Gilardoni MGL 300/6-D X-ray apparatus. After addition of

colcemid and incubation for 90 min, cells were used to prepare chromosome spreads on slides.

After Giemsa staining, chromatid and chromosome breaks, rings and dicentric chromosomes on 50

cells were scored by two operators for each treatment. GTG banding was performed to evaluate

clonal rearrangements involving chromosomes 7 and 14. The colony survival assay and colony

efficiency calculations were performed as reported (35), on LCLs seeded into 96-well tissue culture

plates (100, 200 and 400 cells/well), irradiated with 0 or 1Gy, cultured for 12 days and stained with

MTT; the colonies containing >32 cells were scored positive by optical microscopy. Experiments

were performed in quadruplicate plates.

Western blot analysis

Cells were washed with PBS plus 0.1mM Na3VO4 (Sigma), pelleted and lysed in Laemli buffer

(0.125M Tris-HCl pH 6.8, 5% SDS) containing protease and phosphatase inhibitors. After boiling



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for 2min and sonication, lysates were quantitated by Bradford assay. Aliquotes containing 40µg/ml

of protein plus 5% beta-mercaptoethanol were size-fractionated on 7-10% SDS-PAGE and

electroblotted onto PVDF membranes. After blocking with 5% non-fat dried milk in PBS plus 0.1%

Tween 20, membranes were incubated with monoclonal antibodies for ATM (clone 4D2, made in

house) and Chk2 (clone 44D4/21, made in house) (36), and with rabbit antibodies specific for

NBS1 and Mre11 (GeneTex, San Antonio, TX), p53 phospho-S15 (Cell Signaling Technology,

Beverly, MA), ATM phospho-S1981 (Rockland Immunochemicals, PA), Smc1 phospho-S966

(Bethyl Laboratories, Inc, Montgomery, TX), total Smc1 (Bethyl Laboratories) and β-actin (Sigma,

Italy). Blots tested for ATM phospho-S1981 were reprobed with the monoclonal antibody MAT3-

4G10/8 (37) to normalize for total amounts of ATM. Immunoreactive bands were visualised by

ECL Super Signal (Pierce, Rockford, IL) on autoradiographic films, scanned and quantitated by

ImageQuant software (Molecular Dynamics).

PTT analysis and DNA sequencing

PTT analysis of Mre11 full-length protein was performed as reported (18), using TNT T7 Coupled

Reticulocyte Lysate System (Promega). The cDNA was obtained by RT-PCR from lymphoblastoid

cells, treated or untreated for 4 hours with 0.1mM anisomycin. The PCR conditions were 35 cycles

of denaturation at 94°C for 10s, annealing at 57°C for 30s and extension at 68°C for 2min using

Expand Long template Taq polymerase (Roche). The in vitro translated products were separated on

8% SDS-PAGE and visualized by autoradiography.

The cDNA from anisomycin-treated cells as well as genomic DNA were sequenced for MRE11gene

as reported (18). Briefly, PCR products were purified and 50ng sequenced in 20µl reactions

containing 2µl ABI Prism Big Dye Terminator (PE biosystems), 6µl buffer and 1pmol primer. The

PCR conditions were 25 cycles of 96°C 10s, 50°C 5s, 60°C 4min. Sequencing reactions were

subsequently ethanol precipitated and then resuspended in formamide/dye (5:1) loading buffer.

Samples were run on the ABI Prism 377 DNA sequencer according to ABI protocols.



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Cell cycle analysis

Radiation-induced cell cycle phase modifications were examined by flow cytofluorimetry on

propidium iodide stained cells (34) using a FACSCalibur instrument fitted with a Cell Quest

software package (Becton Dickinson, Sunnyvale, CA).

Ionizing Radiation Induced Foci (IRIF)

Unirradiated cells or irradiated with 12Gy were incubated for 8hrs, cytocentrifuged onto glass slides

and air-dried. After fixation with 4% formaldehyde for 10 min, slides were washed twice in PBS,

permeabilized with 0.2% Triton X-100 for 10 min, blocked with 10% normal goat serum and

incubated overnight with 1:100 dilution of a rabbit antibody to human Mre11 (GeneTex). Following

washing and incubation for 30 min with 1:50 dilution of fluorescein-conjugated anti-rabbit IgG

(Jackson ImmunoResearch, West Grove, PA), slides were washed again and counterstained with

Hoechst 33258. Foci were analysed with an Axioskop 2 Plus fluorescence microscope equipped

with an AxioCam digital camera (Zeiss, Germany). At least 100 nuclei were scored for each

preparation, and were considered positive if contained at least 5 distinct foci.



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Acknowledgements

The Authors wish to thank Prof. A.M. Taylor, The University of Birmingham, Cancer Research UK

Institute for Cancer Studies, the Medical School Edghaston, Birmingham, UK for providing the

ATLD2 and ATLD4 cells, and for sharing preliminary results. Prof. Yossi Shiloh, Department of

Human Genetics and Molecular Medicine Sackler School of Medicine, Tel Aviv University, kindly

provided the anti-ATM monoclonal antibody. This work was financed by grants of the Italian

Telethon Foundation (grants E764 and GP0205Y01), Italian Association for Cancer Research

(AIRC), Consiglio Nazionale Ricerche (CNR grant CU03.00416), the Italian Ministries of Health

and of University and Research (FIRB grant RBNE01RNN7).



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Legends to Figures

Fig 1: Mutation analysis. Electropherograms (A) of MRE11 exon 15 from ATLD5, identifying the

maternal 1442 C→A and paternal 1714 C→T alleles. The latter mutation can be seen in patient’s

genomic DNA, as well as in the cDNA derived from anisomycin-treated cells. (B), schematic

sequence of Mre11 protein (from Ref 12) and position of residues that are affected by the mutations

in ATLD cases. The vertical hatched lines indicate the DNA binding regions a and b.

Fig 2: Radiosensitivity in ATLD cells. Colony survival assays performed on LCLs derived from a

normal donor (LBC-N) and from patients with A-T and ATLD. Cells were irradiated with 1Gy and

cultured for 12 days and analysed for the presence of colonies after MTT staining. The values are

means of three experiments.

Fig 3: Defective expression of Mre11, Rad 50 and Nbs1 proteins in ATLD. Whole cell extracts

prepared from LCLs were tested in western blots with antibodies specific for ATM, Mre11, Rad50

and Nbs1 proteins. Blots were reprobed for β-Actin to normalize lanes for protein content.

Fig 4: Mre11 Nuclear Foci. The immunofluorescence labelling for Mre11 was carried out 8 hours

after exposure of LCLs to 0 or 12Gy of irradiation. Note the formation of radiation-induced Mre11

nuclear foci in LBC-N, but not in ATLD5.

Fig 5: ATM activation and phosphorylation of Chk2. Whole cell extracts prepared from the

indicated LCLs or fibroblasts were tested on western blots with antibodies specific for ATM

pS1981 and for total Chk2. In A, the LCLs were harvested 30min after treatment with 0, 0.25 and

1Gy of IR. Note the difference in ATM autophosphorylation signal, particularly at 0.25Gy, between

LBC-N and ATLD cases. The lack of pS1981 signal in AT52RM cells verified the specificity of the

antibody. Blots were tested with an anti-ATM antibody to verify the total amounts of ATM per



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lane. Chk2 phosphorylation was evaluated on LCLs (B) or primary fibroblasts (FB-ATLD5 and FB-

ATLD6) (C) harvested 30min and 3hrs after 10Gy of IR. Note the absence of a phosphorylation-

related mobility delay in ATLD2, FB-ATLD5 and FB-ATLD6.

Fig 6: Radiation-induced p53-S15 phosphorylation levels. Western blot analysis of p53 and p53-

pSer15 performed on LCLs collected 30 min after exposure to 0 or 10Gy of IR (above). The cells

were pre-incubated with the proteasome inhibitor LLnL to allow the accumulation of basal p53. The

hitsogram (below) displays the leves of p53-pS15, normalized for total p53, obtained by the

densitometric analysis of the western blots.

Fig 7: Radiation-induced Smc1-S966 phosphorylation. Western blot analysis for phosphorylated

Smc1-S966 and for total Smc1 performed on normal, A-T and ATLD LCLs harvested 30 or 60min

after treatment with 0 or 10Gy of IR.

Fig 8: Flow cytofluorimetric analysis of cell cycle phases changes after irradiation.

Lymphoblastoid cells were harvested 24 hrs after exposure to 0 or 10Gy IR, stained with propidium

iodide and analysed by flow cytofluorimetry. The relatively high increase of the G2 peak in

irradiated ATLD5 and ATLD6 is indicative of a G1 checkpoint arrest defect.



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Table 1 – G1/G2-M ratio determined on LCLs by DNA flow cytofluorimetry 24hrs after exposure

to 0 or 10Gy of IR.

Cell line G1/G2-M untr.

G1/G2-M 10Gy 24h

LBC-N 3.2±0.25 2.18±0.53

ATLD2 3 0.9

ATLD4 5.2 1.8

ATLD5 2.8±0.16 0.86±0.15

ATLD6 2.82±0.1 0.77±0.11

AT52RM 3.6 0.65



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Fig 4

LBC-N

ATLD5

Untr. IR



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Fig 7

ATLD

5

LBC-

N

ATLD

6

LBC-

N

ATLD

5AT

LD6

AT52

RM

LBC-N

- -+ - - - - -+ + + + + +Smc1

Smc1-pS966

Smc1

0 6030 0 6030 0 6030

ATLD2 ATLD4Smc1-pS966

IR

A

B

30min 60min

Time, min



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Fig 8

LBC-N ATLD5 ATLD6 ATLD4 AT52RM

Untr.

IR



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Abbreviations

A-T Ataxia-Telangiectasia

ATLD Ataxia-Telangiectasia Like Disorder

ATM Mutated in Ataxia-Telangiectasia

ATR Ataxia-Telangiectasia and Rad3 related

DSBs Double Strand Breaks

EMG Electromyography

GTG G banding with Trypsin and Giemsa staining

IR Ionizing Radiation

LCL Lymphoblastoid Cell Line

LLnL N-acetyl-L-leucinyl-L-leucinyl-norleucinal

MRN Mre11 / Rad50 / Nbs1 complex

MTT 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyl tetrazolium bromide

NBS Nijmegen Breakage Syndrome

NMD Nonsense Mediated mRNA Decay

PBS Phosphate Buffer Saline

PCR Polymerase Chain Reaction

PTT Protein Truncation Test

SDS Sodium Dodecyl Sulphate PAGE Polyacrylamide gel electrophoresis



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Documents

MRE11 mutations and impaired ATM-dependent responses in an Italian family with ataxia-telangiectasia-like disorder