Attenuation of the formation of DNA-repair foci ... - Oxford Academic

Carcinogenesis vol.23 no.7 pp.1121–1126, 2002

Attenuation of the formation of DNA-repair foci containing RAD51 in Fanconi anaemia

Martin Digweed1,6, Susanne Rothe1, Ilja Demuth1, Regina Scholz2, Detlev Schindler3, Markus Stumm4, Markus Grompe5, Andreas Jordan2 and Karl Sperling1 1Institut

fu¨r Humangenetik, Charite´ – Campus Virchow-Klinikum, Humboldt Universita¨t zu Berlin, 2Klinik fu¨r Strahlenheilkunde, Charite´ – Campus Virchow-Klinikum, Humboldt Universita¨t zu Berlin, 3Institut fu¨r Humangenetik, Theodor-Boveri-Institut fu¨r Biowissenschaften (Biozentrum), Bayerische Julius-Maximilians-Universita¨t Wu¨rzburg, 4Institut fu¨r Humangenetik, Universita¨tsklinikum, Medizinische Fakulta¨t der Otto-vonGuericke Universita¨t Magdeburg, Germany and 5Department of Molecular and Medical Genetics, Oregon Health Sciences University, Portland, Oregon, USA 6To

whom correspondence should be addressed at: Institut fu¨r Humangenetik, Charite´ – Campus Virchow, Augustenburger Platz 1, 13353 Berlin, Germany Email: [email protected]

The role of the Fanconi anaemia genes in DNA repair was examined by a quantitative analysis of nuclear DNA repair foci in FA primary fibroblasts after ionising irradiation using antibodies directed against RAD51, MRE11 and BRCA1 for visualisation. IR induced foci detected with anti-RAD51, but not those detected with anti-MRE11, are reduced in fibroblasts of all eight FA complementation groups in comparison to control cells. Correction of FA-A, FA-C and FA-G cells by retroviral cDNA transfer specifically corrected the RAD51-foci response but did not affect formation of foci containing BRCA1 or MRE11. Since all FA cells, except FA-D1, lack the monoubiquitinated FANCD2-L protein, this isoform is likely to be involved in the formation of nuclear foci containing RAD51 in diploid FA cells. FA-D1 cells show the same attenuation in RAD51 foci formation, suggesting that the unknown FANCD1 protein is similarly involved in RAD51 foci formation, either independently or as a subsequent step in the FANCD2 pathway. These findings indicate that Fanconi anaemia cells have an impairment in the RAD51-dependent homologous recombination pathway for DNA repair, explaining their chromosomal instability and extreme sensitivity to DNA cross-linking agents. Introduction The autosomal recessive disorder Fanconi anaemia (FA [MIM 227650]) is characterised clinically by progressive bone marrow failure and an increased cancer risk sometimes associated with various somatic abnormalities. The spontaneous chromosomal breakage seen in patient cells and exacerbated after their exposure to DNA cross-linking agents suggests that defective DNA repair is a major consequence of the FA gene mutations. This characteristic sensitivity of FA cells towards cross-linking agents (1) is accompanied by a much weaker Abbreviations: DSB, double strand break; EGFP, enhanced green fluorescence protein; FA, Fanconi anemia; IR, ionising radiation; PBS, phosphatebuffered saline. © Oxford University Press

and clearly more variable sensitivity to the lesions caused by ionising radiation (2–8). The major lesion associated with ionising radiation (IR) is the DNA double strand break (DSB) for which multiple repairpathways in mammalian cells exist (9,10). DSBs are also thought to be produced during the repair of interstrand crosslinks explaining the occurrence of mutant rodent cells with sensitivity to both IR and DNA cross-linkers (11,12), the reported IR sensitivity of cross-linker sensitive FA cells and, finally, the modest cross-linker sensitivity reported for some IR-sensitive cells from Nijmegen Breakage Syndrome patients (13–15). The two major mechanisms for DSB repair are nonhomologous end joining (NHEJ) and homologous recombination (HR) although the relative usage of these two pathways differs between yeast and mammalian cells (for reviews see refs 16,17). Many genes involved in these two processes have been identified and include RAD51 (HR), MRE11 (NHEJ and HR) and DNA-PKCS (NHEJ). Some of the proteins encoded by these genes accumulate at the sites of DSBs and are visible as discrete nuclear foci by immunofluorescence staining after IR (18–21). The FA protein, FANCD2, also forms nuclear foci in response to IR (22). FANCD2 can be linked to both the HR and NHEJ pathways via BRCA1, a DNA binding protein (23), which is part of a supercomplex, BASC, implicated in genome surveillance (24). Thus, BRCA1 modulates the nuclease activity of MRE11 (23), forms common nuclear foci after IR with RAD51 (25,26) and interacts with FANCD2 in response to DNA damage, also in nuclear foci (22). The role of BRCA1 in the chromosomal instability syndromes including FA has been reviewed recently in ref. 27. FA is genetically heterogeneous with eight complementation groups so far established (FA-A – FA-G) and thus eight causative genes expected (28,29) six of which have been isolated. FANCD2, the gene mutated in FA complementation group FA-D2, was recently identified (30) and is unusual amongst the FA genes in as much as it is conserved in lower eukaryotes and its product is modified by monoubiquitination after DNA damage, a process for which the remaining FA genes, except FANCD1, are required (22). Thus, the unmodified protein FANCD2-S (short) is present in all FA cells, except FA-D2, whilst the modified FANCD2-L (long) is found after clastogenic treatment only in FA-D1 and control cells. The direct interaction of the known FA proteins had made a common pathway likely (31) and indeed several are found together as a multimeric complex (32–34). Here we have examined the IR induced accumulation of nuclear foci in primary fibroblasts from all eight FA complementation groups, and in retrovirally corrected FA-A, FA-C and FA-G cells. For detection of foci we used antibodies directed against RAD51, MRE11 and BRCA1. The results show that the components of nuclear DNA repair foci are influenced by the presence of FANCD2-L and FANCD1 and that these proteins are both required for the efficient 1121

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accumulation of RAD51, but not BRCA1 or MRE11, at the sites of DNA damage. Materials and methods Cell culture and cell irradiation Primary fibroblasts from FA patients were: FA5BER, STT (FA-A); HSC1199 (FA-B); RNT, HRA (FA-C); HSC62 (FA-D1); PD20, PD24, PD733, HND, HNK (FA-D2); FA13BER (FA-E), FA4BER (FA-F); FA15BER, GNF (FA-G). Control cells, LN8 and LN9, were from clinically healthy individuals. All primary fibroblasts were grown in Eagle’s Minimal Essential Medium with 10% foetal bovine serum in 5% CO2 and 10% O2. For some irradiation experiments, cell cycle parameters were monitored by bromodeoxyuridine (BrdU) incorporation and anti-BrdU immunofluorescent staining (Roche) to measure the proportion of cells in S-phase. In some experiments, cells were arrested in G1 by contact inhibition at confluence and serum deprivation. Irradiation of cells grown in 8-chamber microscope-slide flasks (BioCoat, Becton Dickinson) was carried out using the X-ray apparatus Muller MG 150 (UA ⫽ 100 kV, I ⫽ 10 mA, filter 0.3 mm Ni, dose rate: 2.1 Gy/min). Doses ranging from 0 to 12 Gy were applied additively under normal air conditions at 37°C on a thermostated waterbed. Retroviral transduction of FA-C and FA-G cells The MSCV2.1-based retroviral vectors (35) used were LFAPEG expressing the FANCA open reading frame from the 5⬘-LTR, LFCPEG containing the FANCC cDNA, and LFGPEG with the FANCG cDNA. In addition, these vectors carry the gene for enhanced green fluorescence protein (EGFP) behind a second, internal, murine phosphoglycerokinase promoter (Clontech). Retroviral transduction of fibroblast cells was carried out by exposure at intermediate confluence in 6-well plates to viral supernatants. Three rounds of transduction were performed in the presence of 7.5 µg/ml protamine (Roche). Cells were grown for at least two days after the final transduction to ensure stable expression of the cDNAs from integrated proviruses. Transduction efficiency was measured by flow cytometry of the co-expressed EGFP. Immunofluorescence staining and microscopy For indirect immunofluorescence microscopy, cells were fixed for 10 min in 4% paraformaldehyde and permeabilised for 5 min at 4°C in 0.5% Triton X-100. The slides were blocked by incubation overnight at 4°C in 2% BSA in phosphate-buffered saline (PBS). Rabbit polyclonal anti-RAD51, antiMRE11 (Novus Biologicals) and mouse monoclonal anti-BRCA1 (Oncogene Research Products) were used at appropriate dilutions. Incubation with the primary antibodies was for 1 h at 37°C. Carbocyanine (Cy2) or indocarbocyanin (Cy3) conjugated secondary antibodies (Dianova) were used at a dilution of 1:400 and incubation was for 1 h at 37°C. Cell nuclei were counterstained with 4’,6-diamidino-2-phenylindole (DAPI). Digital microscopy was performed with the Zeiss Axiophot microscope equipped with a CCD camera (SensiCam) using the Zeiss filter set 13 (excitation 470, emission 505–530) for Cy2 stains and filter set 20 (excitation 546, emission 575–640) for Cy3 stains. Fluorescent signals were pseudo-coloured by the AxioVision software and optimised for contrast. For foci quantification the slides were coded and 250 or 500 nuclei assessed for the presence of foci using the DAPI stain to count total nuclei. We used no threshold for foci number per nucleus; examples of counted foci can be seen in Figure 1. Results from multiple countings were compared using the unpaired Student’s t-test. In the graphic presentations mean percentages of positive cells are given together with error bars indicating the standard error of the mean. Western blotting Whole cell extracts were prepared by cell lysis in RIPA buffer and were separated on 8% polyacrylamide SDS gels. Proteins were electroblotted to nitrocellulose membranes, blocked in 5% dried milk in TBST and probed with rabbit polyclonal anti-RAD51 and anti-MRE11 (Novus Biologicals) overnight at 4°C followed by incubation with horseradish peroxidaseconjugated secondary antibody diluted 1:1000. Chemiluminescent detection was performed using the ECL reagents (Amersham).

Results RAD51-foci formation is reduced in primary FA fibroblasts but not MRE11-foci formation Figure 1 shows IR induced nuclear foci detected with an antiRAD51 antibody and a Cy3-conjugated secondary antibody in control and representative FA fibroblasts. RAD51-foci are clearly produced after ionising irradiation in both the control 1122

Fig. 1. RAD51 nuclear foci observed in irradiated control and FA cells. Primary fibroblasts from controls and from FA patients were irradiated with 12 Gy and 8 h later were fixed, permeabilised and incubated with a primary rabbit antibody directed towards RAD51 followed by detection with a secondary Cy3-conjugated goat anti rabbit-Ig. Cells were counterstained with DAPI and examined microscopically using the appropriate filters. Representative digital images for two controls (LN8 and LN9), FA-A (FA5BER), FA-D1 (HSC62), FA-D2 (PD20) and FA-G (FA15BER) cells are shown.

and FA cells, however, the foci-quantification shown in Figure 2A shows a reduction in the proportion of cells displaying foci in primary fibroblasts from all FA complementation groups. Unirradiated log phase control and FA cells all show low levels of foci-presenting cells, presumably reflecting cells repairing endogenous DSBs such as those occurring during DNA replication. In contrast, the response to 12 Gy IR is strikingly different. Whilst control cells show a 15-fold increase in foci-presenting cells, FA cells from all complementation groups show, on average, a modest 2-fold increase. Student’s t-test is highly significant (P ⫽ 0.00001) when the percentage of foci presenting nuclei for all FA cells (last column in Figure 2A) is compared to foci presentation in control cells. The difference in foci-presentation of unirradiated control and FA cells, fails to reach significance, indicating that RAD51 expression as such is not reduced in FA cells (see also below). Comparably low levels of foci formation are found in irradiated control and FA cells arrested in G1 by serum starvation, and thus not executing RAD51-mediated HR repair (Figure 2A, grey columns). In contrast, IR induced formation of foci containing MRE11 is unperturbed in primary fibroblasts from all FA complementation groups, as shown in Figure 2B. Cells arrested in G1 by serum starvation produce the same levels of MRE11foci (grey columns in Figure 2B) indicating that NHEJ, unlike HR is not dependent on DNA replication or the presence of sister chromatids for DSB repair. RAD51-foci formation is restored in FA-A, FA-C and FA-G fibroblasts corrected by cDNA transfer To examine the specificity of the reduction in RAD51 in nuclear foci, FA-A, FA-C and FA-G primary fibroblasts were

DNA-repair foci in Fanconi anaemia

Fig. 2. Quantification of RAD51 and MRE11 foci in control and FA fibroblasts. The levels of RAD51 foci-positive cells (A) and MRE11 foci-positive cells (B) are shown for control fibroblasts and fibroblasts from all known FA complementation groups. Open columns represent unirradiated cells, filled columns are cells irradiated with 12 Gy and processed for immunofluorescence 8 h later. Pooled results for FA cells from all complementation groups are shown on the right of the figures as ‘pool’. The fibroblasts were in logarithmic growth, however, cells were also irradiated after arrest in G1 (grey columns) in order to measure foci formation independently of DNA replication. 500 cells/point were counted with duplicate data points.

infected with either an EGFP-expressing retroviral vector or vectors containing, in addition, the FANCA, FANCC or FANCG cDNAs. Examination of co-expressed EGFP fluorescence allowed the efficiency of transduction to be established as 57% for STT (FA-A), 95% for RNT (FA-C) and 79% for GNF (FA-G) transductions. In addition to RAD51 and MRE11, antibodies directed against BRCA1 were also used for foci detection. All of these proteins are known to relocate to nuclear foci after ionising irradiation of cells. Figure 3 shows the quantification of nuclei in irradiated cells displaying foci stained with these three antibodies. Again, MRE11 foci formation is undisturbed in FA-A, FA-C and FA-G primary fibroblasts, as shown above. Consequently, transduction with the FANCA, FANCC and FANCG cDNAs has no effect on MRE11 foci formation. Similarly, BRCA1 foci are as abundant in irradiated FA-A, FA-C and FA-G cells as in control cells, as previously found (22). We observe a slight increase in BRCA1 foci formation in FA-C and FA-G cells transduced with the appropriated cDNAs, however the effects are not statistically significant. Irradiated FA-A, FA-C and FA-G cells in these experiments show again significantly fewer RAD51 foci in comparison to control cells (control:pooled FA cells, P ⫽ 0.00007) and this is clearly not affected by transduction with the empty retroviral vector. All three FA cell strains were specifically corrected in RAD51 foci formation, however, by

Fig. 3. Levels of MRE11, BRCA1 and RAD51-foci formation in corrected FA-A, FA-C and FA-G fibroblasts. FA-A, FA-C and FA-G primary fibroblasts were transduced with empty EGFP retroviral vectors (⫹ vector) or vectors additionally carrying the appropriate FA-cDNA (⫹FANCA; ⫹FANCC; ⫹FANCG). FA-A (STT) and FA-C (RNT) cells were each transduced with one vector preparation (LFAPEG-15, LFCPEG-14), FA-G (GNF) cells were independently transduced with two vector preparations (LFGPEG-5 and LFGPEG-28) and stably transformed cells obtained. Cells were stained for MRE11, BRCA1 or RAD51 and foci-positive cells were counted in irradiated (solid columns) and unirradiated parallel cultures (open columns). Results for control cells (LN8) are also shown. Cells were irradiated with 12 Gy, and 250 cells/point were counted.

the appropriate cDNA (empty vector:cDNA, P ⫽ 0.009 for FA-A; P ⫽ 0.031 for FA-C; P ⫽ 0.002 for FA-G). The response of these cells to 12 Gy irradiation in terms of focipresenting cells is now comparable with that of control cells (control:pooled corrected FA cells, t-test not significant, P ⫽ 0.894). In order to rule out that the effect on RAD51 foci-formation after 12 Gy IR is merely a reflection of the cell cycle, parallel cultures of FA-G fibroblasts, transduced with the empty vector and two independent FANCG-transduced cultures were labelled with BrdU immediately before mock irradiation and the proportion of S-phase cells assessed by immunofluorescent staining with anti-BrdU antibody. The data in Table I show that although the proportion of S-phase cells is lower in uncorrected FA-G cells this cannot explain the paucity of RAD51 foci after irradiation. As the ratio of foci presenting cells in the irradiated cultures to S-phase cells in the mockirradiated cultures clearly shows, what is abnormal in the irradiated, uncorrected FA-G cells is the level of RAD51-foci formation. 1123

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Fig. 4. Western blot of RAD51 in transduced FA-A and FA-G fibroblasts. Cell lysates from control cells (LN9), FA-A fibroblasts (STT) and FA-G fibroblasts (GNF) transduced with empty retroviral vectors or vectors containing FANCA or FANCG were separated by PAGE, blotted to nitrocellulose and probed successively for RAD51 and MRE11 (as a control for gel loading). There are no significant differences in RAD51 expression, in comparison to MRE11, after correction by gene transfer.

Table I. S-phase and RAD51 foci in irradiated control and transduced FA-G cells

Con FA-G ⫹ vector FA-G ⫹ FANCG (I) FA-G ⫹ FANCG (II)

RAD51 (% cells)a

S-phase (% cells)b

Ratio RAD51/ S-phase

18.4 5.4 20.6 23.3

32.3 26.7 34.9 36.9

0.57 0.20 0.59 0.63

aCells

were irradiated with 12 Gy and RAD51 foci presenting cells counted 8 h later. bParallel, mock-irradiated cultures were assessed for S-phase cells at the time of irradiation

Finally, since increased levels of homologous recombination (36) have been reported for FA cells we examined the expression of RAD51 in comparison to MRE11 in corrected and uncorrected FA-A and FA-G fibroblasts. As shown in Figure 4, no significant differences in RAD51 levels are found between uncorrected cells and their retrovirally corrected derivatives, which are normalised in RAD51 foci production. Clearly the attenuation of RAD51 foci formation in FA cells is not a reflection of lower levels of RAD51 expression, at least not in FA-A and FA-G. Discussion The discovery by Garcia-Higuera et al. (22) that the monoubiquitinated isoform of FANCD2, FANCD2-L, associates with BRCA1 and is found in nuclear foci in mutagenised cells has linked the recessive disorder FA to the repair of DNA lesions. Monoubiquitination of FANCD2-S occurs in response to DNA damage and is dependent on the presence of the other FA gene products, except FANCD1, thus coupling them into the DNA-repair response. Our findings on the formation of foci containing RAD51, BRCA1 and MRE11 in FA cells from all eight complementation groups can thus be discussed in relationship to the presence of functional FANCD2-S, FANCD2-L and FANCD1. RAD51-foci formation and Fanconi anaemia proteins RAD51-foci formation after ionising radiation was attenuated in primary fibroblasts of all FA complementation groups and specific retroviral correction of FA-A, FA-C and FA-G cells restored normal levels of RAD51-containing foci. Corrected FA-A, FA-C and FA-G cells are able to modify FANCD2-S to FANCD2-L (22), thus it seems likely that FANCD2-L is 1124

involved in the accumulation of RAD51 at the sites of DNA repair in human cells. Presumably specific correction of cells from the remaining FA complementation groups would also result in normalisation of RAD51 foci-production, although this remains to be demonstrated. RAD51 is the 37 kDa human homologue of bacterial RecA and yeast Rad51 and forms a polymer on DNA in an ATP dependent fashion to facilitate pairing and strand exchange during homologous recombination (37). In addition, mammalian RAD51 is required for cell proliferation and survival (38,39) and is accompanied in the mammalian cell by five paralogues, RAD51B, RAD51C, RAD51D, XRCC2 and XRCC3. All the RAD51 paralogues are involved in homologous recombination (40). The DNA lesion to which FA cells are particularly sensitive is the interstrand cross-link, suggesting that the DNA-repair defect in FA cells is likely to involve homologous recombination. In S.cerevisiae, repair of cross-links utilises homologous recombination and requires DSBs (41,42). In mammalian cells too, repair of DNA cross-links is thought to employ the HR pathway and DSBs (43). Experiments using a cell free assay for DNA cross-link repair demonstrated induction of DNA synthesis both in damaged and in undamaged homologous plasmids, again suggesting that the repair of DNA cross-links occurs by a recombinational mechanism (44). Interestingly, the activity of cell extracts from some FA complementation groups was 50% reduced in this particular study (44). Furthermore, mutations in the RAD51 paralogues in the chicken B-lymphocyte DT40 cell line all result in extreme sensitivity to cross-linking agents, such as Mitomycin C and cisplatin, and also in significantly attenuated RAD51 foci formation after IR (40,45). It has been reported that DSBs can occur at the sites of DNA interstrand cross-links when they are encountered by the replication machinery at a replication fork (46) and other studies have similarly indicated a requirement for DNA replication to elicit cellular responses to DNA interstrand cross-links (47). RAD51 is known to associate with postreplicative chromatin (21). Failure to efficiently recruit RAD51 to the sites of DSBs might explain the anomalies in recovery of DNA-synthesis after cross-linking treatments reported previously for FA cells (4,48–50). The finding, that the FANCA/FANCC/FANCG complex binds to chromatin after DNA-damage (51) suggests a damage sensing function which could instigate DNA repair through monoubiquitination of FANCD2-S and the subsequent interactions of FANCD2-L. DNA repair-foci composition in Fanconi anaemia BRCA1 and several other proteins have been reported to form nuclear foci in response to DNA damage, indicating that large, multimeric complexes are responsible for genome surveillance and repair of DNA lesions (24). Both RAD51 and RAD50/ MRE11/nibrin colocalise in nuclear foci with BRCA1 – however these foci are not necessarily identical, and may be even mutually exclusive. Zhong et al. report that the majority of IR induced BRCA1 foci contain RAD50 whilst only a minority have RAD51; BRCA1-foci containing both RAD51 and RAD50 simultaneously, are not observed (52). Wang et al. have further demonstrated that RAD50 foci are also found completely independently of BRCA1 foci (24). Levels of IR induced foci containing BRCA1 are normal in FA cells as shown here and reported previously (22). Since RAD51 foci are observed, at low levels, in FA cells which

DNA-repair foci in Fanconi anaemia

lack FANCD1, FANCD2-S or FANCD2-L, these proteins are clearly not essential for RAD51 foci formation. However, the significantly reduced numbers of RAD51-foci presenting cells, and the correction of this by cDNA transfer in FA-A, FA-C and FA-G cells, suggests that foci composition is strongly influenced by both FANCD2-L and FANCD1. Neither BRCA1 nor MRE11 shows this dependence on FA proteins. The trimeric complex MRE11/RAD50/nibrin is involved in DSB repair by NHEJ, and in the initial events of repair by HR. The primary FA cells examined here are unaffected in the MRE11/RAD50/nibrin pathway as evidenced by formation of nuclear foci containing MRE11. In agreement with these results, core components of NHEJ have been found to be unaffected in FA-D1 and FA-C cells (5). Furthermore, at DNA:protein ratios which are expected to reflect the physiological situation of repair by DNA-PK-dependent end-joining, no difference in repair of DSBs by nuclear extracts from FA complementation groups A, C and D2 in comparison with control cells was observed (53). The attenuation of RAD51 foci observed in all FA complementation groups, accompanied by unaltered MRE11 foci formation, suggests therefore, that the spectrum of DNA-repair foci produced after IR is influenced by the FA proteins. Thus, FANCD2-L, absent from all but FA-D1 cells, seems to be involved in the association of RAD51 but not MRE11 with BRCA1 in nuclear foci. Interestingly, FA-D1 cells, which do have FANCD2-L, actually show the lowest RAD51 foci formation, indicating that the, as yet unknown, FANCD1 protein acts either independently on RAD51 or in the same pathway between FANCD2-L and RAD51. Irs1, a mutant hamster cell line with mutations in XRCC2 is deficient in DSB repair by homologous recombination (54) and forms no RAD51 foci after IR (55). However, a direct interaction between RAD51 and XRCC2 has not been demonstrated, rather, common binding partners amongst the RAD51 paralogues suggest an indirect association (56). A similar network of interactions may explain the attenuation of RAD51 foci in FA cells lacking FANCD1 or unable to produce FANCD2-L. Interestingly, mouse embryonic stem cells with mutations in the murine homologue of BRCA2, which also associates with RAD51 (57), show no RAD51 foci formation, stimulated repair of DSBs by the RAD51-independent HR pathway of single strand annealing (SSA), and increased chromosomal instability (58). Repair of DSBs by SSA proceeds through the alignment of homologous flanking sequences but does not involve strand exchange and gene-conversion as in RAD51mediated HR. SSA is error prone and results in both deletions and chromosome translocations (59). Undue use of SSA in FA cells might therefore explain the chromatid breaks and chromosome translocations characteristic of FA cells, the increased frequency of genomic deletions after DNA damage (60) and also the finding of increased levels of homologydirected repair in FA cell extracts (36). Note added in proof Recently, Howlett et al. reported that the gene mutated in both FA-B and FAD1 complementation groups is in fact BRCA2 (Science, 13 June 2002, 10.1126/ science, 1073834). The authors propose a model in which transduction signalling to the BRCA2 binding partner, RAD51, is disturbed in FA cells. This model is given experimental support by the findings presented here on RAD51 foci in FA cells.

Acknowledgements We are indebted to Dr Manuel Buchwald, Hospital for Sick Children, Toronto, Canada, for providing us with the FA-B and FA-D1 fibroblasts and to Dr Helmut Hanenberg, Heinrich-Heine-Universita¨ t Du¨ sseldorf, for the FANCA, FANCC and FANCG retroviral vectors. Excellent technical assistance was provided by Gabriele Hildebrand (DNA transfer) and Janina Radszewski (foci quantification), for which we are very grateful. This work was supported by the Fritz-Thyssen-Stiftung (Az 2000/01/69) and the European Union BIOMED2 programme (grant BMH4-CT-98-3784).

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