Transmission ratio distortion of mutations in the master regulator of centriole biogenesis PLK4.

The evolutionary conserved Polo-like kinase 4 (PLK4) is essential for centriole duplication, spindle assembly, and de novo centriole formation. In man, homozygous mutations in PLK4 lead to primary microcephaly, altered PLK4 expression is associated with aneuploidy in human embryos. Here, we report on a consanguineous four-generation family with 8 affected individuals compound heterozygous for a novel missense variant, c.881 T > G, and a deletion of the PLK4 gene. The clinical phenotype of the adult patients is mild compared to individuals with previously described PLK4 mutations. One individual was homozygous for the variant c.881G and phenotypically unaffected. The deletion was inherited by 14 of 16 offspring and thus exhibits transmission ratio distortion (TRD). Moreover, based on the already published families with PLK4 mutations, it could be shown that due to the preferential transmission of the mutant alleles, the number of affected offspring is significantly increased. It is assumed that reduced expression of PLK4 decreases the intrinsically high error rate of the first cell divisions after fertilization, increases the number of viable embryos and thus leads to preferential transmission of the deleted/mutated alleles.

Directed Alternative Splicing in Nijmegen Breakage Syndrome: Proof of Principle Concerning Its Therapeutical Application.

Over 90% of patients with Nijmegen breakage syndrome (NBS), a hereditary cancer disorder, are homoallelic for a 5 bp deletion in the NBN gene involved in the cellular response to DNA damage. This hypomorphic mutation leads to a carboxy-terminal protein fragment, p70-nibrin, with some residual function. Average age at malignancy, typically lymphoma, is 9.7 years. NBS patients are hypersensitive to chemotherapeutic and radiotherapeutic treatments, thus prevention of cancer development is of particular importance. Expression of an internally deleted NBN protein, p80-nibrin, has been previously shown to be associated with a milder cellular phenotype and absence of cancer in a 62-year-old NBS patient. Here we show that cells from this patient, unlike other NBS patients, have DNA replication and origin firing rates comparable to control cells. We used here antisense oligonucleotides to enforce alternative splicing in NBS patient cells and efficiently generate the same internally deleted p80-nibrin protein. Injecting the same antisense sequences as morpholino oligomers (VivoMorpholinos) into the tail vein of a humanized NBS murine mouse model also led to efficient alternative splicing in vivo. Thus, proof of principle for the use of antisense oligonucleotides as a potential cancer prophylaxis has been demonstrated.

DNA damage in Nijmegen Breakage Syndrome cells leads to PARP hyperactivation and increased oxidative stress.

Nijmegen Breakage Syndrome (NBS), an autosomal recessive genetic instability syndrome, is caused by hypomorphic mutation of the NBN gene, which codes for the protein nibrin. Nibrin is an integral member of the MRE11/RAD50/NBN (MRN) complex essential for processing DNA double-strand breaks. Cardinal features of NBS are immunodeficiency and an extremely high incidence of hematological malignancies. Recent studies in conditional null mutant mice have indicated disturbances in redox homeostasis due to impaired DSB processing. Clearly this could contribute to DNA damage, chromosomal instability, and cancer occurrence. Here we show, in the complete absence of nibrin in null mutant mouse cells, high levels of reactive oxygen species several hours after exposure to a mutagen. We show further that NBS patient cells, which unlike mouse null mutant cells have a truncated nibrin protein, also have high levels of reactive oxygen after DNA damage and that this increased oxidative stress is caused by depletion of NAD+ due to hyperactivation of the strand-break sensor, Poly(ADP-ribose) polymerase. Both hyperactivation of Poly(ADP-ribose) polymerase and increased ROS levels were reversed by use of a specific Poly(ADP-ribose) polymerase inhibitor. The extremely high incidence of malignancy among NBS patients is the result of the combination of a primary DSB repair deficiency with secondary oxidative DNA damage.

The nuclease hSNM1B/Apollo is linked to the Fanconi anemia pathway via its interaction with FANCP/SLX4.

The recessive genetic disorder Fanconi anemia (FA) is clinically characterized by congenital defects, bone marrow failure and an increased incidence of cancer. Cells derived from FA patients exhibit hypersensitivity to DNA interstrand crosslink (ICL)-inducing agents. We have earlier reported a similar cellular phenotype for human cells depleted of hSNM1B/Apollo (siRNA). In fact, hSNM1B/Apollo has a dual role in the DNA damage response and in generation and maintenance of telomeres, the latter function involving interaction with the shelterin protein TRF2. Here we find that ectopically expressed hSNM1B/Apollo co-immunoprecipitates with SLX4, a protein recently identified as a new FA protein, FANCP, and known to interact with several structure-specific nucleases. As shown by immunofluorescence analysis, FANCP/SLX4 depletion (siRNA) resulted in a significant reduction of hSNM1B/Apollo nuclear foci, supporting the functional relevance of this new protein interaction. Interestingly, as an additional consequence of FANCP/SLX4 depletion, we found a reduction of cellular TRF2, in line with its telomere-related function. Finally, analysis of human cells following double knockdown of hSNM1B/Apollo and FANCP/SLX4 indicated that they function epistatically. These findings further substantiate the role of hSNM1B/Apollo in a downstream step of the FA pathway during the repair of DNA ICLs.

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.

An essential function for NBS1 in the prevention of ataxia and cerebellar defects.

Nijmegen breakage syndrome (NBS), ataxia telangiectasia and ataxia telangiectasia-like disorder (ATLD) show overlapping phenotypes such as growth retardation, microcephaly, cerebellar developmental defects and ataxia. However, the molecular pathogenesis of these neurological defects remains elusive. Here we show that inactivation of the Nbn gene (also known as Nbs1) in mouse neural tissues results in a combination of the neurological anomalies characteristic of NBS, ataxia telangiectasia and ATLD, including microcephaly, growth retardation, cerebellar defects and ataxia. Loss of Nbn causes proliferation arrest of granule cell progenitors and apoptosis of postmitotic neurons in the cerebellum. Furthermore, Nbn-deficient neuroprogenitors show proliferation defects (but not increased apoptosis) and contain more chromosomal breaks, which are accompanied by ataxia telangiectasia mutated protein (ATM)-mediated p53 activation. Notably, depletion of p53 substantially rescues the neurological defects of Nbn mutant mice. This study gives insight into the physiological function of NBS1 (the Nbn gene product) and the function of the DNA damage response in the neurological anomalies of NBS, ataxia telangiectasia and ATLD.

New mutations in the ATM gene and clinical data of 25 AT patients.

Ataxia telangiectasia (AT) is an autosomal recessive disorder characterized by cerebellar degeneration, immunodeficiency, oculocutaneous telangiectasias, chromosomal instability, radiosensitivity, and cancer predisposition. The gene mutated in the patients, ATM, encodes a member of the phosphatidylinositol 3-kinase family proteins. The ATM protein has a key role in the cellular response to DNA damage. Truncating and splice site mutations in ATM have been found in most patients with the classical AT phenotype. Here we report of our extensive ATM mutation screening on 25 AT patients from 19 families of different ethnic origin. Previously unknown mutations were identified in six patients including a new homozygous missense mutation, c.8110T>C (p.Cys2704Arg), in a severely affected patient. Comprehensive clinical data are presented for all patients described here along with data on ATM function generated by analysis of cell lines established from a subset of the patients.

Mutations in the telomerase component NHP2 cause the premature ageing syndrome dyskeratosis congenita.

Dyskeratosis congenita is a premature aging syndrome characterized by muco-cutaneous features and a range of other abnormalities, including early greying, dental loss, osteoporosis, and malignancy. Dyskeratosis congenita cells age prematurely and have very short telomeres. Patients have mutations in genes that encode components of the telomerase complex (dyskerin, TERC, TERT, and NOP10), important in the maintenance of telomeres. Many dyskeratosis congenita patients remain uncharacterized. Here, we describe the analysis of two other proteins, NHP2 and GAR1, that together with dyskerin and NOP10 are key components of telomerase and small nucleolar ribonucleoprotein (snoRNP) complexes. We have identified previously uncharacterized NHP2 mutations that can cause autosomal recessive dyskeratosis congenita but have not found any GAR1 mutations. Patients with NHP2 mutations, in common with patients bearing dyskerin and NOP10 mutations had short telomeres and low TERC levels. SiRNA-mediated knockdown of NHP2 in human cells led to low TERC levels, but this reduction was not observed after GAR1 knockdown. These findings suggest that, in human cells, GAR1 has a different impact on the accumulation of TERC compared with dyskerin, NOP10, and NHP2. Most of the mutations so far identified in patients with classical dyskeratosis congenita impact either directly or indirectly on the stability of RNAs. In keeping with this effect, patients with dyskerin, NOP10, and now NHP2 mutations have all been shown to have low levels of telomerase RNA in their peripheral blood, providing direct evidence of their role in telomere maintenance in humans.

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.

DNA repair 2008: Tenth Biennial Meeting of the German Society for Research on DNA Repair.

The Tenth Meeting of the German Society for Research on DNA Repair was held in Berlin in September 2008. Invited presentations by Yosef Shiloh, Stanton L. Gerson, Sacha Beneke, Patrick Concannon, Jochen Dahm-Daphi, Thilo Dörk, Friedrike Eckardt-Schupp, Bernd Epe, Ian Hickson, Ulrich Hübscher, Penny Jeggo, Malik Lutzmann, Christof Niehrs, Primo Schär and Predrag Slijepcevic together with over 80 selected oral and poster presentations generated an inspiring scientific program, which documented the impressive progress of the community and defined future challenges in the field.

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.

Endogenous hSNM1B/Apollo interacts with TRF2 and stimulates ATM in response to ionizing radiation.

Human SNM1B/Apollo is involved in the cellular response to DNA-damage, however, its precise role is unknown. Recent reports have implicated hSNM1B in the protection of telomeres. We have found hSNM1B to interact with TRF2, a protein which functions in telomere protection and in an early response to ionizing radiation. Here we show that endogenous hSNM1B forms foci which colocalize at telomeres with TRF1 and TRF2. However, we observed that additional hSNM1B foci could be induced upon exposure to ionizing radiation (IR). In live-cell-imaging experiments, hSNM1B localized to photo-induced double-strand breaks (DSBs) within 10s post-induction. Further supporting a role for hSNM1B in the early stages of the cellular response to DSBs, we observed that autophosphorylation of ATM, as well as the phosphorylation of ATM target proteins in response to IR, was attenuated in cells depleted of hSNM1B. These observations suggest an important role for hSNM1B in the response to IR damage, a role that may be, in part, upstream of the central player in maintenance of genome integrity, ATM.

Extreme variation in apoptosis capacity amongst lymphoid cells of Nijmegen breakage syndrome patients.

The human genetic disorder, Nijmegen breakage syndrome (NBS), is characterised by radiosensitivity, immunodeficiency and an increased risk for cancer, particularly lymphoma. The NBS1 gene codes for a protein, nibrin, involved in the processing/repair of DNA double strand breaks and in cell cycle checkpoints. The majority of patients (>90%) are homozygous for a founder mutation. Despite this genetic homogeneity, the syndrome shows considerable clinical variability, for example, in age at development of a malignancy. We hypothesised that one reason for such variation might be individual differences in the clearance of heavily damaged precancerous cells by apoptosis. To test this hypothesis we have examined a set of 30 lymphoblastoid B-cell lines from NBS patients for their capacity to enter into apoptosis after a DNA-damaging treatment. There was a substantial 40-fold variation in apoptosis between cell lines from different patients. NBS patient cell lines could be grouped into a large, apoptosis-deficient group and a smaller group with essentially normal apoptotic response to DNA damage. In both groups, cell lines were proficient in TP53 phosphorylation and stabilisation after the same DNA-damaging treatment. Thus the observed variation in apoptosis capacity is not due to failure to activate TP53. Despite the large variation in apoptosis, no statistically significant correlation between apoptotic capacity of patient cell lines and clinical course of the disease was apparent.

Yeast XRS2 and human NBN gene: Experimental evidence for homology using codon optimized cDNA.

The genes, XRS2 in Saccharomyces cerevisiae and NBN in mammals, have little sequence identity at the amino acid level. Nevertheless, they are both found together with MRE11 and RAD50 in a highly conserved protein complex which functions in the repair of DNA double-strand breaks. Here, we have examined the evolutionary and functional relationship of these two genes by cross-complementation experiments. These experiments necessitated sequence correction for specific codon usage before they could be successfully conducted. We present evidence that despite extreme sequence divergence nibrin can, at least partially, replace Xrs2 in the cellular DNA damage response, and Xrs2 is able to promote nuclear localization of MRE11 in NBS cells. We discuss that the extreme sequence divergence reflects a unique adaptive pressure during evolution related to the specific eukaryotic role for both Xrs2 and nibrin in the subcellular localisation of the DNA repair complex. This, we suggest, is of particular relevance when cells are infected by viruses. The conflict hypothesis of co-evolution of DNA repair genes and DNA viruses may thus explain the very low sequence identity of these two homologous genes.

Somatic mutations and progressive monosomy modify SAMD9-related phenotypes in humans.

It is well established that somatic genomic changes can influence phenotypes in cancer, but the role of adaptive changes in developmental disorders is less well understood. Here we have used next-generation sequencing approaches to identify de novo heterozygous mutations in sterile α motif domain-containing protein 9 (SAMD9, located on chromosome 7q21.2) in 8 children with a multisystem disorder termed MIRAGE syndrome that is characterized by intrauterine growth restriction (IUGR) with gonadal, adrenal, and bone marrow failure, predisposition to infections, and high mortality. These mutations result in gain of function of the growth repressor product SAMD9. Progressive loss of mutated SAMD9 through the development of monosomy 7 (-7), deletions of 7q (7q-), and secondary somatic loss-of-function (nonsense and frameshift) mutations in SAMD9 rescued the growth-restricting effects of mutant SAMD9 proteins in bone marrow and was associated with increased length of survival. However, 2 patients with -7 and 7q- developed myelodysplastic syndrome, most likely due to haploinsufficiency of related 7q21.2 genes. Taken together, these findings provide strong evidence that progressive somatic changes can occur in specific tissues and can subsequently modify disease phenotype and influence survival. Such tissue-specific adaptability may be a more common mechanism modifying the expression of human genetic conditions than is currently recognized.

Nbn heterozygosity renders mice susceptible to tumor formation and ionizing radiation-induced tumorigenesis.

Nijmegen Breakage Syndrome (NBS) is a rare autosomal recessive disease characterized by microcephaly, growth retardation, immunodeficiency, chromosomal instability, and predisposition to cancer. Heterozygous NBS patients show increased chromosomal instability and are suspected to be at a high risk for cancer. To study the impact of NBS1 heterozygosity on malignancy susceptibility, we disrupted the murine homologue (Nbn) of NBS1 in mice using gene targeting techniques. While null mutation in the Nbn gene resulted in embryonic lethality at the blastocyst stage because of growth retardation and increased apoptosis, heterozygous knockout (Nbn(+/-)) mice developed a wide array of tumors affecting the liver, mammary gland, prostate, and lung, in addition to lymphomas. Moreover, gamma-irradiation enhanced tumor development in Nbn(+/-) mice, giving rise to a high frequency of epithelial tumors, mostly in the thyroid and lung, as well as lymphomas. These mice also developed numerous tumors in the ovary and testis. Southern and Western blot analyses showed a remaining wild-type allele and nibrin expression in Nbn(+/-) tumors. Sequencing analysis confirmed no mutation in the Nbn cDNA derived from these tumors. Cytogenetic analysis revealed that primary Nbn(+/-) embryonic fibroblasts and tumor cells exhibit increased chromosomal aberrations. These data suggest that haploinsufficiency, not loss of heterozygosity, of Nbn could be the mechanism underlying the tumor development. Taken together, our heterozygous Nbn-knockout mice represent a novel model to study the consequences of NBS1 heterozygosity on tumor development.

The Slavic NBN Founder Mutation: A Role for Reproductive Fitness?

The vast majority of patients with Nijmegen Breakage Syndrome (NBS) are of Slavic origin and carry a deleterious deletion (c.657del5; rs587776650) in the NBN gene on chromosome 8q21. This mutation is essentially confined to Slavic populations and may thus be considered a Slavic founder mutation. Notably, not a single parenthood of a homozygous c.657del5 carrier has been reported to date, while heterozygous carriers do reproduce but have an increased cancer risk. These observations seem to conflict with the considerable carrier frequency of c.657del5 of 0.5% to 1% as observed in different Slavic populations because deleterious mutations would be eliminated quite rapidly by purifying selection. Therefore, we propose that heterozygous c.657del5 carriers have increased reproductive success, i.e., that the mutation confers heterozygote advantage. In fact, in our cohort study of the reproductive history of 24 NBS pedigrees from the Czech Republic, we observed that female carriers gave birth to more children on average than female non-carriers, while no such reproductive differences were observed for males. We also estimate that c.657del5 likely occurred less than 300 generations ago, thus supporting the view that the original mutation predated the historic split and subsequent spread of the 'Slavic people'. We surmise that the higher fertility of female c.657del5 carriers reflects a lower miscarriage rate in these women, thereby reflecting the role of the NBN gene product, nibrin, in the repair of DNA double strand breaks and their processing in immune gene rearrangements, telomere maintenance, and meiotic recombination, akin to the previously described role of the DNA repair genes BRCA1 and BRCA2.

Human SNM1B is required for normal cellular response to both DNA interstrand crosslink-inducing agents and ionizing radiation.

DNA interstrand crosslinks (ICLs) are critical lesions for the mammalian cell since they affect both DNA strands and block transcription and replication. The repair of ICLs in the mammalian cell involves components of different repair pathways such as nucleotide-excision repair and the double-strand break/homologous recombination repair pathways. However, the mechanistic details of mammalian ICL repair have not been fully delineated. We describe here the complete coding sequence and the genomic organization of hSNM1B, one of at least three human homologs of the Saccharomyces cerevisiae PSO2 gene. Depletion of hSNM1B by RNA interference rendered cells hypersensitive to ICL-inducing agents. This requirement for hSNM1B in the cellular response to ICL has been hypothesized before but never experimentally verified. In addition, siRNA knockdown of hSNM1B rendered cells sensitive to ionizing radiation, suggesting the possibility of hSNM1B involvement in homologous recombination repair of double-strand breaks arising as intermediates of ICL repair. Monoubiquitination of FANCD2, a key step in the FANC/BRCA pathway, is not affected in hSNM1B-depleted HeLa cells, indicating that hSNM1B is probably not a part of the Fanconi anemia core complex. Nonetheless, similarities in the phenotype of hSNM1B-depleted cells and cultured cells from patients suffering from Fanconi anemia make hSNM1B a candidate for one of the as yet unidentified Fanconi anemia genes not involved in monoubiquitination of FANCD2.

SV40 large T-antigen disturbs the formation of nuclear DNA-repair foci containing MRE11.

The accumulation of DNA repair proteins at the sites of DNA damage can be visualized in mutagenized cells at the single cell level as discrete nuclear foci by immunofluorescent staining. Formation of nuclear foci in irradiated human fibroblasts, as detected by antibodies directed against the DNA repair protein MRE11, is significantly disturbed by the presence of the viral oncogene, SV40 large T-antigen. The attenuation of foci formation was found in both T-antigen immortalized cells and in cells transiently expressing T-antigen, indicating that it is not attributable to secondary mutations but to T-antigen expression itself. ATM-mediated nibrin phosphorylation was not altered, thus the disturbance of MRE11 foci formation by T-antigen is independent of this event. The decrease in MRE11 foci was particularly pronounced in T-antigen immortalized cells from the Fanconi anaemia complementation group FA-D2. FA-D2 cells produce essentially no MRE11 DNA repair foci after ionizing irradiation and have a significantly increased cellular radiosensitivity at low radiation doses. The gene mutated in FA-D2 cells, FANCD2, codes for a protein which also locates to nuclear foci and may, therefore, be involved in MRE11 foci formation, at least in T-antigen immortalized cells. This finding possibly links Fanconi anaemia proteins to the frequently reported increased sensitivity of Fanconi anaemia cells to transformation by SV40. From a practical stand point these findings are particularly relevant to the many studies on DNA repair which exploit the advantages of SV40 immortalized cell lines. The interference of T-antigen with DNA repair processes, as demonstrated here, should be borne in mind when interpreting such studies.