Human Molecular Genetics, 2002, Vol. 11, No. 21 2531-2546
© 2002 Oxford University Press
DNA cross-link-dependent RAD50/MRE11/NBS1 subnuclear assembly requires the Fanconi anemia C protein
1UPR 2169–Institut André Lwoff IFR 2249 CNRS-7, rue Guy Môquet, 94801 Villejuif Cedex, France and 2UMR 2027 CNRS/IC–Institut Curie Recherche–Centre Universitaire, Bâtiment 110, 91405 Orsay Cedex, France
Received March 25, 2002; Accepted July 11, 2002
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Fanconi anemia (FA) is a cancer-predisposition syndrome characterized by hypersensitivity to interstrand-cross-link (ICL) inducers. FA hypersensitivity to ICL has been correlated with alterations in homologous recombination, non-homologous end-joining, telomere maintenance, DNA-damage assessment and checkpoint regulation, processes in which the components of the RAD50/MRE11/NBS1 (RMN) complex are involved. To better characterize the mechanisms by which ICL are processed in human cells and to gain insight into their toxicity in FA, we examined (i) the RMN complex assembling in response to the ICL inducers mitomycin C (MMC) and photoactivated 8-methoxypsoralen and (ii) the proficiency of FA cells to perform RMN activation in response to ICL inducers. We show here that ICL activates the assembly of the RMN proteins into subnuclear foci, and that their formation proceeds independently of ICL incision, a step mainly dependent on XP-F/ERCC1 heterodimer activity. Interestingly, FA cells were unable to form RMN foci in response to either ICL inducer. Analysis by pulsed-field gel electrophoresis and single-cell gel electrophoresis of MMC-treated cells showed that FA cells from complementation group C (FA-C cells, defective in the FANCC gene) form double-strand breaks and unhook MMC-induced ICL similarly to FANCC wild-type cells. These observations imply that the absence of RMN assembly in FA-C cells is not simply due to the absence of DNA ends produced as intermediates of ICL processing, and indicates a direct role for FANCC in RMN focus assembly in response to ICL inducers. Moreover, we show that the formation of foci, including BRCA1 and/or RAD51 proteins, is significantly delayed in FA cells. These alterations in the assembly of DNA-repair proteins in FA provide an interpretation for the DNA-damage processing anomalies observed in FA cells and for the genetic instability and the cancer predisposition of the syndrome.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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DNA interstrand cross-links (ICL) are lesions introduced into DNA by many carcinogens and antitumor drugs such as photoactivated psoralens, cisplatin, mitomycin C (MMC) and derivatives of nitrogen mustard. Since ICL act as a block for DNA and RNA polymerases, their presence is highly toxic for cells. Studies in Escherichia coli and yeast indicate that both nucleotide excision repair (NER) and homologous recombination (HR) are required for ICL removal (1). In addition to the intervention of the HR pathway, a potential role for non-homologous end-joining (NHEJ) in ICL repair has been reported (2). Finally, since an mre11 strain shows ICL sensitivity and a defect in ICL-associated double-strand break (DSB) rejoining similar to that of the HR-defective strain rad52, a key role for Mre11 in ICL repair was suggested (2). Mre11 forms a complex with Rad50 and Xrs2 (called the RMX complex) that is involved in both HR and NHEJ (3).
The isolation and characterization of mutant cell lines with extreme sensitivities to cross-linking agents provide evidence for a combined intervention of NER and recombination in ICL repair also in mammalian cells (1). In particular, a central role was suggested for the NER excinuclease heterodimer XPF–ERCC1 (4–6). Moreover, the involvement of XPF–ERCC1 in ICL repair has been clearly supported by work describing the biochemical steps through which this heterodimer contributes to ICL removal (7,8). Studies with cell lines disrupted for components of the HR system as well as the extreme hypersensitivity to ICL agents of the cells mutated in XRCC2 and XRCC3 (paralogs of RAD51) clearly indicate the importance of HR in resolving ICL also in mammals (9–11). This pathway of ICL removal is specific to S and G2 phases of the cell cycle. The NHEJ may influence the sensitivity of mammalian cells to ICL-inducing drugs (12). However, compared with the ERCC1/XPF and HR mutants, NHEJ-deficient cells show only a moderate sensitivity to ICL agents. This suggests that this pathway plays a minor role in ICL repair. Finally, the involvement of mammalian MRE11, alone or as a component of the RAD50/MRE11/NBS1 (RMN) complex, structurally and functionally homologous to the yeast RMX complex, remains unexplored.
A particular class of human ICL-hypersensitive mutants comprises the cells carrying mutations in one of the Fanconi anemia (FA) genes. FA is a rare human autosomal recessive syndrome featuring aplastic anemia, cancer predisposition, genetic instability, hypersensitivity to exposure to ICL agents and, to a lesser extent, to ionizing radiations (13). Six of the eight responsible genes, FANCA, FANCC, FANCD2, FANCE, FANCF and FANCG, have been cloned (14–20). They have little, if any, homology to each other or to other known genes. Although the functions of the FA proteins are still unknown, they may assemble in complex(es) that appear(s) to cooperate in common cellular pathways. Conflicting data suggest that the FA proteins may function in several processes, including metabolism of reactive oxygen species, cell cycle checkpoints, apoptosis and DNA repair (13,21). Several lines of evidence suggest that FA cells have an underlying molecular defect in DNA repair. In particular, HR, NHEJ, telomere maintenance DNA-damage assessment and checkpoint regulation have been reported as altered in FA (22–26). However, despite extensive analysis of this defect, the biochemical events under the control of the FA proteins as well as any direct involvement of these proteins in ICL repair remain elusive (21).
As studies in yeast indicate an important role for Mre11 in ICL removal (2) and the RMN protein complex is known to play a pivotal role in the same molecular processes that are described as altered in FA cells [namely HR, NHEJ, telomere maintenance, DNA-damage assessment and checkpoint regulation (3,27–29)], we decided to analyse the functionality of this complex in FA cells.
In this study, we show that ICL activates the aggregation of the RMN proteins into bright-punctuated structures, termed foci, inside the cell nucleus. Evidence is presented for the first time that FA protein activity is required for the assembly of the RMN complex into such subnuclear structures specifically in response to MMC-induced ICL. It is shown that the RMN complex requires broken DNA extremities to be assembled. During ICL processing, DNA breaks are produced as repair intermediates during the HR and the NER steps of the process. Analysis by pulsed-field gel electrophoresis (PFGE) and single-cell gel electrophoresis (SCGE) of MMC-treated cells showed that FA-C cells form double-strand breaks and unhook MMC-induced ICL, similarly to cells expressing wild-type FA complex. This demonstrates that the absence of RMN assembly in FA-C cells is not simply due to the absence of broken DNA ends, and indicates a direct role for FANCC in RMN foci assembly in response to exposure to an ICL inducer. Moreover, as a possible consequence of the absence of RMN focalization, the formation of foci, including BRCA1 and/or RAD51 proteins, is significantly delayed in FA cells. These alterations in the assembly of DNA-repair proteins in FA provide an interpretation for the S/G2 phase and DNA-damage processing anomalies observed in FA cells, and would largely contribute to the genetic instability of the syndrome. Taken together, our results support a pivotal role for the RMN complex in DNA-repair processes and cell cycle checkpoint control, extending its activity to ICL removal also in mammals.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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MMC-induced RAD50/MRE11/NBS1 focus formation is defective in FA cells
To investigate whether FANCC function is required for the subnuclear assembly of the RMN complex following DNA damage, we used an isogenic pair of cell lines, with two inactive FANCC alleles (HSC-536) or overexpressing the wild-type FANCC gene (HSC-536CORR). Two normal cell lines, HSC-93 and SNW646, were also included in our analysis. As reported, the MMC dose-dependent inhibition of growth in the FA-C cell line HSC-536 is significantly higher than in HSC-536CORR and HSC-93 cells (Fig. 1). For example, exposure to 0.1 µg/ml/1 h of MMC inhibits growth by 60% in FA-C cells, versus 10% in corrected and normal cells, demonstrating the hypersensitivity of FA cells to the cytotoxic and cytostatic effect of the ICL-inducer drug.
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To study RMN subnuclear assembly, cells were fixed and then labelled using anti-MRE11 and anti-NBS1 antibodies, and their nuclei were analyzed by confocal microscopy (Fig. 2A). The correct co-localization of each of the three partners of the RMN complex has been verified in MRE11/NBS1 and NBS1/RAD50 double-labelling experiments, ensuring that the NBS1 and RME11 nuclear foci are representative of the RMN complex (data not shown). In response to the ICL inducer MMC (0.1 µg/mg/1 h) normal B lymphoblasts displayed an increase in terms of both RMN-positive nuclei and the number of RMN foci per nucleus (Fig. 2A, a–f; Fig. 2B, a and b). The formation of the foci in normal cells was time-dependent, reaching the maximum at 6 hours and resuming the basal level 15 hours after treatment. In sharp contrast, we failed to observe formation of NBS1 or MRE11 foci in response to MMC treatment in the FA-C cell line HSC-536 (Fig. 2A, g–l; Fig. 2B, a and b). In MMC-treated HSC-536 cells, NBS1 and MRE11 showed a diffuse pattern of labelling, similar to the pattern observed in untreated cells (Fig. 2A). A similar result, i.e. absence of RMN foci formation, was also observed in FA cells exposed to two other doses of MMC (0.01 or 1.0 µg/ml/1 h) (data not shown). Ectopic overexpression of wild-type FANCC in HSC-536 cells allowed recovery of the focus-forming activity of NBS1 and MRE11 after MMC (Fig. 2A, p–u; Fig. 2B, a and b), suggesting that MMC-dependent subnuclear assembly of these proteins requires a functional FANCC protein. To further investigate the role of FA in RMN assembly, we analyzed NBS1 and MRE11 focalization in EUFA143L, an FA-G cell line (Fig. 2B, a and b) and in HSC-72, an FA-A cell line (data not shown). Both cell lines showed the same inability to form RMN nuclear foci as HSC-536 cells. Altogether, these results demonstrate that RMN focalization in response to MMC requires FA proteins from different complementation groups, and consequently depends on the activity of the FA protein complex.
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In response to ionizing radiation, which induces mainly DNA strand breaks, FANCC-/- cells formed RMN nuclear foci similarly to normal and HSC-536CORR cell lines (Fig. 2A, m–o, Fig. 2B, C and D and data not shown). Thus, the presence of an active FA complex is necessary for the correct subnuclear assembly of the RMN complex only after treatment with an ICL inducer.
It has previously been reported that NBS1 protein undergoes ATM/ATR-dependent phosphorylation (30–34). Therefore, to better define the defect in RMN activation in FA, we next analyzed NBS1 phosphorylation on proteins extracted from HSC-536, HSC-536CORR and SNW646 lymphoblasts exposed to MMC or 
-rays. Treatment with ionizing radiation resulted in a slowly migrating form of NBS1 protein in normal cells as well as in FANCC-/- and its corrected counterpart (Fig. 3A). In contrast, in response to MMC, the slowly migrating form of NBS1 appeared in normal and FA-C corrected cells, but not in FANCC-/- lymphoblasts (Fig. 3A). The mobility of the slowly migrating form of NBS1 from MMC-treated HSC536CORR cells reverted to that of NBS1 from untreated cells upon incubation with phosphatase (Fig. 3B). Treatment with phosphatase had no effect on proteins extracted from treated HSC-536 cells, demonstrating the absence of post-translational modifications of NBS1 in FA-C cells (Fig. 3B). These results indicate that NBS1 becomes phosphorylated in response to MMC and that FANCC activity is required for the MMC-induced post-translational modification of NBS1. Our data also showed no difference in the expression levels of NBS1, MRE11 or RAD50 in wild-type, FA and FA complemented cells, either untreated or treated with -rays or MMC (Fig. 3A and C, and data not shown). Therefore, while the FA proteins are not involved in the RMN protein expression, our results suggest that an active FA complex acts prior to both RMN subnuclear assembly and NBS1 phosphorylation.
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To further validate our observations, we analyzed RMN focus formation in primary fibroblasts from a normal donor and from FA-A, FA-C and FA-G complementation groups patients. As seen in Figure 4A and B, primary FA fibroblasts exposed to cross-links induced by photoactivated 8-methoxypsoralen (8-MOP+UVA) or MMC showed the same defect in RMN focalization observed in Epstein–Barr Virus (EBV)-transformed B lymphoblasts exposed to MMC. Using an FA-G-derived SV40-immortalized fibroblastoid cell line and its derived ectopically corrected cell line, we demonstrated that ectopic correction of the FA genotype allowed recovery of the focus-forming activity of RMN after 8-MOP+UVA (Fig. 4A and B). Moreover, treatment with MMC or 8-MOP+UVA resulted in a slowly migrating form of NBS1 protein in normal and FANCG corrected fibroblasts, but not in parental FA-G cells (Fig. 4C).
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MMC exposure induces DNA double-strand breaks in FA and corrected cells
It has been clearly demonstrated in eukaryotic cells that ICL processing involves the formation of DSB (4). The impairment of RMN complex formation in FA might simply be due to the absence of DSB intermediates during the repair of ICL. To investigate if this is the case, exponentially growing lymphoblasts were exposed to MMC, and DSB formation was assessed by PFGE (Fig. 5A). The migration of the DNA into the gel evaluated by PFGE, i.e. the fraction of DNA released from plugs into the gel (FAR), is proportional to the presence of DSB. It is interesting to note the formation of a low-molecular-weight band (
50 kb) of DNA in the gel, particularly evident at 24 hours' recovery. Accordingly to previous studies (35,36), this fraction is probably of apoptotic origin due to the high MMC dose [1 µg/ml/1 h (data not shown) or 10 µg/ml/h] used to consistently show the release of high-molecular-weight DNA (>1 Mb) from plugs into the gel. If only this fraction of DNA released from plugs into the gel is taken into account for the semiquantitative analysis of the FAR, it appears clearly that, after exposure to MMC, DSB accumulate up to 6 hours post treatment and are resorbed after 24 hours in both FA-C and FA-C corrected cells (Fig. 5B). Therefore, DSB are produced after MMC exposure in both FANCC-defective and FANCC-corrected cells.
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These data demonstrate that the absence of RMN complex assembly in FA cells is not a mere consequence of DSB absence in these cells, and suggest direct involvement of FA complex activity in the subnuclear assembly of the RMN complex after exposure to MMC.
Unhooking of MMC-induced ICL is normally achieved in FA cells
The repair of ICL also involves the formation of DNA single-strand breaks as a consequence of ICL incision. So, two questions arise: (i) Are FA-C cells able to incise ICL? (ii) Is the ICL incision step necessary for assembly of the RMN complex?
To investigate whether MMC sensitivity and the defective RMN assembly in MMC-treated FA cells were due to alterations in the incision step of ICL repair, the unhooking efficiency of MMC-induced ICL was analysed in FA-C cells. ICL induction and unhooking was evaluated at single-cell level using the SCGE (comet) assay. The alkaline version of this assay allows evaluation of the presence of single- and double-strand breaks in DNA. The extent of migration of the DNA from the cell nucleus during electrophoresis is directly proportional to the number of breaks induced. To quantify the level of induced ICL, the classical protocol of the comet assay was slightly modified (37). MMC-treated cells were irradiated with 5 Gy before lysis to induce random DNA strand breakage. The presence of ICL retards the migration of the irradiated, broken, DNA during electrophoresis, resulting in a reduced tail moment (TM) in MMC-treated cells compared with that of the MMC-untreated irradiated controls. The reduction in TM is proportional to the level of induced ICL. Figure 6A shows the MMC-dose-dependent decreases in TM in the three cell lines examined. The reduction in TM due to the presence of ICL in FA-C cells was similar to that of the control cell lines. This indicated that exposure to equimolar doses of MMC induces a similar level of ICL, independently of the presence of an active FANCC gene product.
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Analysis of the reduction of the irradiation-induced TM 24 hours after the MMC pulse permitted evaluation of the ability of the treated cells to process ICL. Figure 6B reports the unhooking efficiency of HSC-536 FA-C cells compared with those of the HSC-536CORR and HSC-93 cell lines. The FA-C cell line showed a dose-dependent unhooking indistinguishable from those of corrected and normal cell lines. All three cell lines examined were able to unhook
50% of the 1 µg/ml/h MMC-induced ICL by 24 hours. Similar results were also obtained in cells treated with 8-MOP+UVA, emphasizing that intrastrand cross-links are normally incised in FA-C cells (data not shown).
It has previously been demonstrated that XP-F and ERCC1 mutants, which are extremely sensitive to exposure to ICL agents, were highly defective in the unhooking of ICL but were able to form DSB normally in response to an ICL inducer (4). Consequently, to further characterize the pathway leading to ICL-dependent RMN assembly, we took advantage of the cells deprived of wild-type XP-F activity. The kinetics and the quantity of MRE11 foci assembled in response to MMC in the XP-F cells were similar to what was observed in the control cell line used (Fig. 7).
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Altogether, these data clearly show that the MMC sensitivity as well as the defective RMN assembly of FA-C cells are not due to a defect in ICL unhooking, and that RMN assembly does not require ICL incision.
MMC-induced RAD51 focus-forming activity is altered in FA cells
Our observations demonstrated that, in spite of the formation of DNA broken ends during ICL processing in FA-C cells, the RMN complex is not assembled in the absence of FANCC activity, suggesting a direct role for this protein and probably for the FA complex in mediating RMN assembly. It has been proposed that the RAD50/MRE11/NBS1 complex acts, at least in part, to produce a single-stranded DNA tail onto which other proteins are loaded to complete the repair process, and to signal the damage to other molecular complexes (3,29,34,38). Among these proteins, RAD51, essential for the homology search and the strand invasion steps of the HR pathway, plays a role in ICL removal (2). Therefore, to better define the consequences of the RMN assembly deficiency in FA cells, we decided to analyze also RAD51 focus-forming activity in FA cells.
To study RAD51 subnuclear assembly, cells were fixed and then labelled using an anti-RAD51 antibody, and their nuclei were analyzed by confocal microscopy (Fig. 8A). Approximately 5% of FANCC-/- untreated cells showed RAD51 foci. In both HSC-536CORR and normal cells, the frequency of RAD51-positive cells was slightly but constantly higher (10%). Exposure of the two normal cell lines to MMC (0.1 µg/ml/1 h) induced an increase in terms of both RAD51-positive nuclei and the number of RAD51-foci per nucleus. This increase was time-dependent, reaching its highest value at 6–8 hours' recovery, resuming the basal level 24 hours post treatment (Fig. 8B, a and b). In contrast, in response to MMC treatment, HSC-536 cells displayed a reduced, delayed and more prolonged RAD51 focus-formation activity (Fig. 8B, a and b). Correction of the FANCC-/- cell line with the wild-type FANCC gene permitted complete recovery of RAD51 subnuclear assembly after MMC-induced DNA damage (Fig. 8B, a and b). The same defect in the kinetics of RAD51 assembly observed in the FA-C HSC-536 cell line was also observed in the FA-G EUFA143L cell line (Fig. 8B, a and b) and in the FA-A cell line HSC-72 (data not shown).
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It is possible that the defect in the MMC-dependent subnuclear assembly of RAD51 proteins in FA might be due to excessive accumulation of FA cells in G1 phase in the presence of ICL. We therefore performed flow-cytometric analysis in HSC-93, HSC-536 and HSC-536CORR cell lines at 8 and 24 hours' of recovery. As shown in Figure 8C, the fraction of cells in G1 phase was lower in FANCC-/- than in normal and HSC-536CORR cells. Thus, the delay in the formation of RAD51 foci in FA cells cannot be due to an accumulation of the cells in G1 phase in response to MMC, and cannot be explained simply as an effect of perturbations in the progress through the cell cycle.
In response to ionizing radiation (5 Gy) 8 hours after treatment, the frequency of RAD51-positive cells is slightly reduced in FA compared with the cells expressing wild-type FA genes (Fig. 8B, c). However, the kinetics of assembly and disassembly of the foci as a function of the time and the number of RAD51 foci per nucleus were similar in all tested cell lines (Fig. 8B, c and d). Flow-cytometric analysis of cells 8 and 24 hours after ionizing radiation showed that the fraction of cells in G1 phase was higher in HSC-536 cells compared with HSC-536CORR cells (Fig. 8C). This might account for the difference in the fraction of cells that failed to show foci 8 hours after irradiation.
As evaluated by western blot analysis, both steady-state and genotoxic treatment-dependent RAD51 protein levels were similar in HSC-536, HSC-536CORR and SNW646 cells (Fig. 8D). This suggests that the defect in the kinetics of RAD51 focus formation observed in FA cells after MMC exposure is not due to a defect in protein expression.
MMC-dependent BRCA1 focus formation is delayed in FA cells
It has recently been reported that in response to DNA damage FANCA, -B, -C, -E and -F and BRCA1 are necessary for both the monoubiquitination and the subnuclear assembly of FANCD2 (39). Interestingly, it was shown that BRCA1 may interact and co-localize with either RMN or RAD51 complexes in response to exposure to DNA-damaging agents (40–42). These reasons prompt us to investigate the MMC-induced subnuclear assembly of BRCA1 protein, comparing HSC-536 cells with HSC-536CORR and SNW646 normal cells. Lymphoblasts were treated with MMC (0.1 µg/ml/1 h) and allowed to recover for 4, 8 or 24 hours. Samples were fixed and double-stained using antibodies against BRCA1 and RAD51 (Fig. 9A).
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Consistently with other reports (42,43), BRCA1 foci were found in untreated nuclei. The similar basal frequency of BRCA1-positive nuclei (
20%) observed between the three cell lines used (Fig. 9B) suggests that FANCC is not essential for the subnuclear assembly of BRCA1 in untreated cells. After MMC treatment, an increase in the percentage of BRCA1-positive nuclei was observed in normal and HSC-536CORR cells at 8 and 24 hours of recovery (Fig. 9B). In contrast, the number of FANCC-/- cells showing BRCA1 foci remained near the basal level up to 8 hours. A level similar to that of the other cell lines was then observed 24 hours post treatment. In HSC-536 as in normal cells, BRCA1 co-localizes with RAD51 (Fig. 9B), supporting the fact that, although under altered kinetics, FA cells are proficient in the assembling of these proteins. Like NBS1, BRCA1 protein was phosphorylated in response to DNA damage (44–47). As shown in Fig. 9, FANCC-/- and its corrected counterpart displayed a similar level and phosphorylation of BRCA1. Consequently, the delay in BRCA1 focus formation in FA cells after MMC is not a result of a failure in protein expression, nor is it due to a lack of BRCA1 phosphorylation.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Our present work demonstrates that the assembly of the RMN complex into subnuclear foci is involved in the response to the ICL inducers MMC and photoactivated 8-MOP in human cells. Moreover, using FA cells, which are hypersensititive to ICL agents, we show here that FANCC and the FA protein complex are required for the MMC-dependent assembly of the RMN complex and for the correct temporal subnuclear relocalization of both BRCA1 protein and RAD51 recombinase, both of which are involved in HR. ICL repair involves both NER and recombination pathways (1). The XPF/ERCC1 heterodimer in combination with the HR system are the major players involved in ICL removal, mainly in replicating cells. In yeast, at least one of the component of the RM(X)N complex, involved in both HR and NHEJ, MRE11, has been reported to be involved in ICL repair (2). In mammals, the roles of MRE11 alone and for the RMN complex in ICL removal are poorly characterized. Although RNA antisense-mediated reduction of RAD50 expression induces hypersensitivity to both MMC and ionizing radiation in human cells (48), the MMC sensitivity of cells from Njimegen breakage syndrome (Nbs), mutated in NBS1, or ataxia telangiectasia-like disorder (A-TLD), mutated in MRE11, has not been extensively analysed. However, it is not surprising that MRE11- or NBS1-defective cells did not recapitulate totally FA hypersensitivity to ICL, which is a consequence of mutations in FA genes that alter several pathways, including that leading to RMN focus formation.
Our work presents several pieces of evidence consistent with the hypothesis that the subnuclear assembly of the RMN complex contributes to the repair of ICL in human cells. First, RMN complex activity is evoked in response to both MMC and photoactivated 8-MOP ICL inducers. Second, FA cells, which are specifically hypersensitive to agents producing ICL (49–51), are deficient in RMN complex assembly in response to both MMC and photoactivated 8-MOP. Finally, the ectopic overexpression of the wild-type FA gene in FA cells corrects both the ICL hypersensitivity of the cells and the deficiency in RMN complex focalization. Thus, our data are novel and potentially important, since they suggest not only a role for the RMN complex in ICL repair but also a link between FA and other genomic instability syndromes, and lead us to propose a mechanistic model for the action of the FA complex in ICL repair (Fig. 10). We can envisage that the FA complex enables formation of RMN foci and, together with BRCA1, activates FANCD2. Subsequently, possibly driven by BRCA2, which is involved in the MMC response (52,53), RAD51 is added to BRCA1 foci. Further studies are under way to test this model.
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FA and RMN complexes in ICL repair
It is known that, in spite of a normal sensitivity to ultraviolet light and to monofunctional alkylating agents, FA cells are unequivocally hypersensitive to alkylating bifunctional agents (49–51) and, in several cases, to ionizing radiation (54,55). The reasons for FA hypersensitivity to ICL agents, i.e. misrepair of induced lesions, altered drug detoxification or altered free-radical metabolism, remain uncertain and controversial (21). Among others, Clarke et al. (56) support the hypothesis that the sensitivity of FA-C cells is due to oxygen radical generation and not to DNA cross-linking. Combining data showing that FA cells are in a pro-oxidant state with the observations that the metabolically activated form of MMC is re-oxidized by molecular oxygen to its inactive state, these authors proposed that in response to MMC, FA cells undergo less DNA ICL and more oxidative damage than FA wild-type cells (56). In sharp contrast to this, using the denaturation–renaturation gel electrophoresis assay (DRGE), Youssoufian (57) reported that FA-C cells' hypersensitivity to MMC is due to a higher initial induction of ICL and not to a defect in repair synthesis. Our data from PFGE and SCGE analysis indicate that FA-C cells are able (i) to induce ICL similarly to normal cells, (ii) to form DSB and (iii) to unhook ICL after MMC exposure similarly to corrected and normal cells. Our data are partially in contrast to those of Youssoufian (57), since the induction of ICL at equimolar doses of MMC was similar in FA and normal cells. This may be due to different sensitivities of the assays used (DRGE versus SCGE) to measure MMC-induced ICL. In particular, as discussed by Youssoufian (57), the assessment of ICL within the rRNA by DRGE assay may be not an accurate reflection of the general susceptibility of the whole genome to ICL-induced toxicity. In line with Youssoufian's data, our observations suggest that FA-C cells are able to efficiently repair MMC-induced DNA damage. Thus, it seems unlikely that the lack of RMN complex assembly observed in FA-C cells is caused by the absence of DNA strand breaks (Fig. 4), the substrate for RMN complex assembly, after MMC treatment, nor is it caused by a lower ICL induction, since the quantity of lesions induced is similar in FA and normal cells (Fig. 5A). Moreover, using 8-MOP+UVA-induced lesions, we have furnished a supplementary demonstration that impaired RMN focus formation likely correlates with ICL formation. It is important to note that FA cells assemble the RMN complex properly in response to either ionizing radiations (a treatment resulting in DNA strand breaks, oxidative damage and replication fork arrest) or hydroxyurea (unpublished results), which essentially induces replication fork stall. Altogether, our observations clearly indicate that FA proteins are involved in RMN assembly in the presence of a specific DNA lesion induced by exposure to ICL agents different from those that are also induced after ionizing radiations or hydroxyurea (i.e. DNA breaks, replication fork stall and oxidative damage). Recently, it has been proposed that the FA complex detects ICL through the association of the spectrin protein (58), and it has been reported that FANCC interacts with the SWI/SNF chromatin remodelling complex (59). Thus, it could be proposed that the FA complex is needed to somehow sense the initial lesion or the aberrant chromatin structure surrounding the ICL to correctly process the lesion. It is important to remember that FA cells show alterations in mutation frequency as well as in the molecular spectrum of the induced mutants after exposure to ICL agents (60,61). These observations indicate that the DNA repair defect in FA may reside in the fidelity of the process, not in the intrinsic ability to remove DNA lesions. In this context, the processing of the repair intermediates becomes of primary importance. Consequently, we propose that in FA cells, in the absence of the RMN complex assembly described here, particular form(s) of DSB induced in response to ICL must rely on error-prone pathway(s) of repair, which probably play a minor role in normal cells, leading to the genetic instability of the syndrome. In accordance with this hypothesis, it has been previously observed that FA cells are unable to end-join specific DSB accurately (26,62). Finally, it has been reported that FA cells have altered NHEJ process (22,24,26) and elevated recombinational activity (23). The absence of RMN assembly demonstrated here might be the missing link connecting these two alterations to one common origin. Indeed (at least in yeast), a failure in the proper assembly of the RMN complex has been reported as being responsible for NHEJ impairment (63) and increased HR (64–66). Finally, since a normal ICL unhooking activity was found in FA cells and a normal assembly of MRE11 foci was observed in XPF cells, it is likely that the FA complex lies upstream and is unnecessary to the excision events during ICL repair.
FA complex and S-phase checkpoint
The RMN complex counteracts genomic instability through its influence on both DNA repair and cell cycle control mechanisms. Consequently, the observed defect in its subnuclear assembly may contribute also to the S/G2 phase abnormalities of the FA cells. It was recently shown that FA cells are deficient in ICL-dependent S-phase checkpoint activation (67,68). This alteration might be responsible for the subsequent accumulation of unrepaired cells in G2 phase (69–71). Our data provide a possible explanation for the cell cycle defect of FA. Indeed, it has been reported that NBS1 is phosphorylated by ATM and that the phosphorylated form participates in the activation of the DNA damage-dependent S-phase arrest (30–34). Therefore, the deficiency in the ICL-dependent NBS1 phosphorylation observed here may be directly responsible for the S-phase checkpoint alteration in FA. Mutations in the ATM, MRE11 and NBS1 genes, from patients with ataxia telangiectasia (AT), A-TLD and Nbs, respectively, abrogate this checkpoint.
FA complex and the HR machinery
Our data also demonstrate that BRCA1 and RAD51, involved in the HR pathway (72), are assembled into nuclear foci under delayed kinetics in MMC-treated FA cells. Interestingly, this delayed kinetics was associated with a longer focus-forming activity of RAD51. This may also contribute to the elevated HR activity detected in FA. It has previously been reported that upon DNA damage, the BRCA1 protein may interact with either RMN or RAD51 (40–42). While the interaction of BRCA1 with the RMN complex results in MRE11 inhibition and displacement from DNA (73), the subsequent interaction of BRCA1 with RAD51 is necessary for the subnuclear assembly and activation of this last protein (41). Interestingly, although RMN foci are found in the same nucleus as RAD51 foci, they do not co-localize (42). This suggests that the interaction of BRCA1 with both RMN and RAD51 is mutually exclusive and that BRCA1 represents a physical and functional link between these two systems. Therefore, it is possible that the absence of the subnuclear assembly of the RMN complex after MMC in FA is responsible for the delay in BRCA1 focalization, which in turn affects RAD51 focus formation.
Conclusions
Our observations establish a functional connection between the FA and RMN proteins. They also suggest that the FA complex confers cellular and chromosomal resistance to ICL agents by permitting the correct subnuclear assembly of the RMN complex. Together with the observations demonstrating a connection between FA and BRCA1 (39), our data bring further evidence on a link between FA and human syndromes featuring cancer predisposition, hypersensitivity to DNA damage and genetic instability, such as hereditary breast cancer, AT, Nbs and A-TLD. Indeed, the gene products mutated in the above-mentioned human pathologies, i.e. BRCA1 and BRCA2, ATM, NBS1, and MRE11, interact physically or functionally with each other or are included in the recently identified BASC complex (42). It will be interesting to know whether FA proteins physically interact with BASC components and to determine the mechanism(s) by which FA proteins regulate RMN subnuclear assembling.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Cell lines and genotoxic treatments
EBV-transformed lymphoblasts from FA-A (HSC-72), FA-C (HSC-536) and FA-G (EUFA143B) or normal (HSC-93 and SNW646) donors, and normal, FA-A, FA-C, FA-G and XP-F primary or immortalized fibroblasts were kindly provided by M. Buchwald (Hospital for Sick Children, Toronto, Ontario, Canada), H. Joenje (Department of Human Genetics, Vrije University, Amsterdam, Netherlands), V. Bohr (NIH, Baltimore, MD, USA), B.A. Cox (Oregon Health and Science University, Fanconi Anemia Cell Repository, Portland, OR, USA) and A. Sarasin (CNRS, Villejuif, France). HSC-536CORR is an FA-C (HSC-536) cell line transduced with wild-type FANCC cDNA. These cells express a high level of wild-type FANCC RNA, as verified by northern blot hybridization (data not shown), and are functionally complemented for MMC hypersensitivity (Fig. 1). HSC-536Neo is an FA-C cell line (HSC-536) transduced with the empty vector carrying the neomycin resistance gene; its sensitivity to MMC is unchanged compared with that of the parental cell line (Fig. 1). All cell lines were routinely maintained in exponential growth in RPMI 1640 medium (Life Technologies, USA) supplemented with 12–15% fetal calf serum (Dutcher, France) by a daily dilution to 3x105 cells/ml. HSC-536Neo and HSC-536CORR cells were grown in the presence of 200 µg/ml of G418 (Sigma, USA).
MMC (Sigma, USA) was added from a stock solution (1 mg/ml in PBS), left in the cultures at 37°C for 1 h and then washed out. Alternatively, cells were exposed to -rays from a Cobalt-60 source at a dose rate of 2 Gy/min. After exposure, cells were cultivated in complete medium at 37°C up to the selected time at which they were processed. In the case of 8-methoxypsoralen (Sigma, USA) plus UVA, cells were treated with 10-5 M 8-MOP in PBS during 15 min in the dark and immediately exposed to 1 kJ/m2 as described previously (74).
Immunofluorescence
Cells recovered for the indicated time were spread onto poly-L-lysine-coated slides, fixed for 10 min at room temperature in a 4% paraformaldehyde-buffered solution, and permeabilized with 0.5% Triton X-100 for 10 min at 4°C. Staining with rabbit polyclonal anti-RAD51 (Oncogene Research, USA), rabbit polyclonal anti-MRE11 (Novus BioLabs, USA), mouse monoclonal anti-MRE11 (GenTex, USA), rabbit polyclonal anti-NBS1 (Novus BioLabs, USA), mouse monoclonal anti-RAD50 (GenTex, USA) or mouse monoclonal anti-BRCA1 (Oncogene Research, USA) was performed overnight at 4°C in PBS/1% BSA, whereas species-specific fluorescein- or Texas Red-conjugated secondary antibodies (Jackson Immunoresearch, USA) were applied for 1 h at room temperature followed by counterstaining for 5 min at room temperature with 0.5 µg/ml DAPI. Slides were mounted in Vectashield (Vector Labs, USA) and analyzed by fluorescence confocal microscopy. All the primary antibodies were used at a 1 : 300 dilution, whereas the secondary antibodies were employed at a 1 : 500 dilution. For the simultaneous visualization of RAD51 and PCNA foci, cells were fixed in -20°C methanol for 20 min, followed by 10 min incubation in an ice-cold 1 : 1 methanol/acetone solution. Cell preparations were blocked for 1 h in 10%FBS/PBS, rinsed, and incubated with primary antibodies for 1 h at 37°C. Rabbit polyclonal anti-RAD51 antibody was diluted 1 : 200, while mouse monoclonal anti-PCNA antibody (Santa Cruz Biotechnology, USA) was diluted to 5 µg/ml. For each time point, at least 200 nuclei were examined, and RAD51, BRCA1 or MRE11 foci were scored at a magnification of 100x using a fluorescence confocal microscope. Only nuclei showing more than five foci were considered as positive. Parallel samples, incubated either with the appropriate normal serum or only with the secondary antibody, confirmed that the observed fluorescence pattern was not attributable to artifacts.
Preparation of cell lysates and western blot analysis
Cells (1x107 cells per sample) were collected by centrifugation, washed in PBS and lysed in standard RIPA buffer (PBS, 1% NP-40, 0.5% sodium dehoxycholate, 0.1% SDS, 10 µg/ml Aprotinin, 10 µg/ml PMSF, 1 mM sodium orthovanadate and 1 mM sodium fluoride). Cell lysates (20 µg) were resolved by SDS–PAGE and transferred to nitrocellulose (PROTRAN, Schleicher & Schuell, USA). Equal loading and transfer was monitored by Ponceau Red staining of the membrane. Phosphatase treatment was performed with 500 U of 
-phosphatase on 100 µg of cell lysate resuspended in -phosphatase buffer (New England Biolab, UK). Blots were separately incubated with primary antibodies against NBS1 (1 : 2000), RAD51 (1 : 1000) or BRCA1 (1 : 100). Goat species-specific secondary antibodies (Santa Cruz Biotechnology, USA) labelled with horseradish peroxidase, were used at dilutions of 1 : 2000. Visualization of the signal was accomplished using ECL (Amersham, USA).
Cell cycle analysis
To evaluate the effect of MMC or ionizing radiation exposure on cell cycle progression, untreated or treated cells were harvested at 8 and 24 h recovery. Thirty minutes before harvesting, cell cultures were pulsed with bromodeoxyuridine (BrdU) (45 µM) to label S-phase cells, fixed in cold ethanol, and subsequently stained for replicative DNA synthesis with a fluorescein isothiocyanate (FITC)-conjugated anti-BrdU antibody and for DNA content with propidium iodide (PI), and analyzed by flow cytometry. The percentage of cells in each cell stage was evaluated using the CellFit software (Becton-Dickinson, USA).
Growth-inhibition assay
For the growth-inhibition assay, aliquots from the same cell culture were exposed to the indicated MMC doses for 1 h at 37°C or left in complete culture medium. After washing with PBS, cells were resuspended in complete medium at 105 cells/ml and seeded in 24-well plates in 1 ml of culture medium in duplicate. After 4 days of growth at 37°C, cells in duplicate wells were pooled and counted in a Coulter counter. Untreated controls were processed in parallel. Each experiment was performed at least four times. Growth inhibition was determined as follows:
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Analysis of MMC-induced DBS by pulsed field gel electrophoresis
Exponentially growing lymphoblasts (3–5x106 cells per sample) were exposed to MMC (1 or 10 µg/ml/h), washed to eliminate the drug, and resuspended in complete medium at 37°C for the indicated time. The preparation of cells for PFGE was done following the procedures previously described (75,76). For PFGE, we used a CHEF Mapper TM with a cooling Module (Mini Chiller Model 1000) (Bio-Rad, USA) containing permanently circulating 1xTAE buffer. We included molecular weight markers of chromosomal DNA of Hansenula wingei and Schizosaccharomyces pombe (Bio-Rad, USA) in PFGE agarose gel. The conditions for the CHEF electrophoresis were 74 h total migration time at 2 V/cm with an angle of reorientation of 53° (106°) and a pulse of 35 min. The temperature was maintained at 14°C by a minichiller (Bio-Rad, USA). To analyze the PFGE gels we used the optical method (75), including staining with 0.8 µg/ml ethidium bromide, visualization of fluorescence in a 313 nm UV transilluminator (Ultraviolet Products Inc., USA) and photography with a Polaroid apparatus (film 55 positive/negative) (Polaroid, USA), followed by analysis of the negatives by an image analyzer with the Lecphor program (BioCom, France). The values for the fraction of activity released (FAR) from the wells into the gel, used as an index for DNA DSB, were determined for each electrophoretic lane using the relationship
 |
where Fwell corresponds to the integral of the gray values for the well and Flane to the integral of gray values for the complete lane (including the well).
Determination of ICL induction and repair by single-cell gel electrophoresis analysis (Comet assay)
SCGE was performed in alkaline conditions as previously reported (77), with minor modifications to evaluate the presence of ICL (37). Briefly, exponentially growing cells were exposed to the reported doses of MMC for 1 h at 37°C. The cell cultures were centrifuged, and the pellet was washed in PBS, resuspended in complete medium, and processed immediately or incubated for 24 h to permit recovery. Cells were embedded in 0.5% low-melting-point agarose (final concentration 104 cells/ml). Seventy-five microliters of this cellular suspension were spread on duplicate frosted microscope slides previously covered with 120 µl of a 0.5% basal agarose layer. A coverslip was added and agarose was allowed to solidify for 15 min on ice. Subsequently, control and MMC-treated cells were irradiated (5 Gy) or left on ice before lysis. For lysis, the coverslips were removed and the slides were placed at 4°C for 1 h in lysis buffer (2.5 M sodium chloride, 0.1 M EDTA, 0.01 M Tris, 1% sodium sarcosinate, 1% Triton X-100 and 10% DMSO, pH 10). After lysis, the slides were transferred for 40 min in alkaline buffer (NaOH 300 mM, EDTA 1 mM, pH 13) in the dark to allow DNA to unwind. The slides were transferred in an electrophoresis apparatus, and electrophoresis was carried out at 0.66 V/cm for 25 min (with the current adjusted to 300 mA) in the dark. After neutralization in 0.4 M Tris, pH 7.4, the slides were stained with ethidium bromide (20 µg/ml) and stored at 4°C until analysis. A minimum of 50 cells was analyzed for each sample with a Colormorph Interactive Image Analysis System (Perceptive Instruments Ltd., UK). Each experiment was performed at least in triplicate.
The degree of DNA ICL present in MMC-treated sample was estimated by comparing the tail moment (TM) of the irradiated drug-treated samples (TMdi) with that of the irradiated untreated samples (TMci) and that of the unirradiated untreated samples (TMcu). The quantity of ICL is proportional to the decrease in TM in the irradiated drug-treated sample compared with the irradiated untreated control. The decrease in TM is calculated using the formula
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The unhooking of DNA ICL was expressed as the percentage of unhooking, which was calculated using the formula
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where T0 is the time at the end of the drug treatment and T1 is the recovery time in drug-free medium.
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ACKNOWLEDGEMENTS |
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The authors thank C. Guillouf for a critical review of the manuscript, and M. Zohair, G. Desauty, D. Rouillard and C. Roullin for excellent technical assistance. This work was supported by grants from Electricité de France (EDF), Association pour la Recherche sur le Cancer (ARC), Fondation de France and GEFLUC. P. P. was supported by a grant from EDF.
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FOOTNOTES |
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* To whom correspondence should be addressed. Tel: +33 149583414; Fax: +33 149583411; rosselli{at}vjf.cnrs.fr
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