The Mre11/Rad50/Nbs (MRN) complex and the two protein kinases ATM and ATR play critical roles in the response to DNA damage and telomere maintenance in mammalian systems. It has been previously shown that mutations in the Drosophila mre11 and rad50 genes cause both telomere fusion and chromosome breakage. Here, we have analyzed the role of the Drosophila nbs gene in telomere protection and the maintenance of chromosome integrity. Larval brain cells of nbs mutants display telomeric associations (TAs) but the frequency of these TAs is lower than in either mre11 or rad50 mutants. Consistently, Rad50 accumulates in the nuclei of wild-type cells but not in those of nbs cells, indicating that Nbs mediates transport of the Mre11/Rad50 complex in the nucleus. Moreover, epistasis analysis revealed that rad50 nbs, tefu (ATM) nbs, and mei-41 (ATR) nbs double mutants have significantly higher frequencies of TAs than either of the corresponding single mutants. This suggests that Nbs and the Mre11/Rad50 complex play partially independent roles in telomere protection and that Nbs functions in both ATR- and ATM-controlled telomere protection pathways. In contrast, analysis of chromosome breakage indicated that the three components of the MRN complex function in a single pathway for the repair of the DNA damage leading to chromosome aberrations.

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THE MRN complex contains the two highly conserved proteins Mre11 and Rad50 and a third less-conserved component, Nbs/Xrs2 (D'AMOURS and JACKSON 2002; ASSENMACHER and HOPFNER 2004). This complex plays critical roles in the response to DNA damage and telomere maintenance in both yeast and mammalian systems (D'ADDA DI FAGAGNA et al. 2004; STRACKER et al. 2004; ZHANG et al. 2005). Hypomorphic mutations in the Nbs and Mre11 genes lead to the Nijmegen breakage syndrome (NBS) and to ataxia telangiectasia-like disorder (ATLD), respectively. NBS and ATLD share common features, including chromosome instability, radiation hypersensitivity, immunological disorders, and cancer predisposition. However, while ATLD is characterized by cerebellar degeneration resulting in ataxia, NBS is characterized by microcephaly and growth retardation (DIGWEED and SPERLING 2004; STRACKER et al. 2004). These clinical differences are likely to reflect functional differences between the Nbs and Mre11 components of the human MRN complex.

The components of the MRN complex have multiple and complex interactions with the two conserved protein kinases ATM (Tel1 in Saccharomyces cerevisiae) and ATR (Mec1 in S. cerevisiae). For example, it has been shown that the mammalian MRN complex acts both upstream and downstream of ATM in the DNA damage response. The complex mediates both ATM activation and ATM kinase activity by facilitating its binding to substrates (UZIEL et al. 2003; LEE and PAULL 2004, 2005; CEROSALETTI et al. 2006). The MRN complex also enhances several ATR-dependent phosphorylation events (STIFF et al. 2005; ZHONG et al. 2005). Moreover, it has been shown that ATM and ATR can phosphorylate the same substrates, including the Nbs protein (reviewed by SHILOH 2003). Finally, there is evidence that the components of the MRN complex can act independently in mediating ATM activation and phosphorylation events (CEROSALETTI and CONCANNON 2004; LEE and PAULL 2004). Mutations in ATM and ATR result in the genetic disorders ataxia telangiectasia (AT) and Seckel syndrome, respectively. AT has stronger but similar clinical features to those of ATLD, while Seckel patients have features that overlap NBS, including pronounced microcephaly (STEWART et al. 1999; GOODSHIP et al. 2000; STRACKER et al. 2004).

Studies in mammalian cells have shown that the ATM and ATR kinases and the MRN complex are required for both chromosome integrity and proper telomere function (PANDITA 2002; SHILOH 2003; D'ADDA DI FAGAGNA et al. 2004; STRACKER et al. 2004; ZHANG et al. 2005). However, although these proteins have been extensively studied at the biochemical level, their functional relationships in the maintenance of chromosome stability have not been determined. Progress in understanding such relationships has been hampered because null mutations in the genes encoding the components of the MRN complex lead to early lethality in vertebrates (XIAO and WEAVER 1997; LUO et al. 1999; ZHU et al. 2001). In contrast, thanks to the maternal effect that characterizes Drosophila development, null mutations in the mre11, rad50, and nbs genes cause lethality at late larval stages, allowing cytological analysis of dividing neuroblasts in larval brains. Previous studies have shown that mutations in the Drosophila mre11, rad50, nbs, and tefu (ATM) genes cause both telomeric fusions and chromosome breakage and that tefu and mei-41 (ATR) control redundant pathways of telomere protection (BI et al. 2004, 2005; CIAPPONI et al. 2004; OIKEMUS et al. 2004; SILVA et al. 2004; SONG et al. 2004). Here, we have explored the role of the Drosophila nbs gene in both telomere protection and the maintenance of chromosome integrity. Our results indicate that the Nbs protein and the Mre11/Rad50 complex make distinct contributions to telomere protection but function in a single pathway to prevent chromosome breakage.

Drosophila strains and crosses:

l(3)67BDp was obtained from the Bloomington Stock Center and rebalanced over TM6C. The rad50^5.1, mre11^DC, tefu^atm6, and mei41^29D mutations have been described previously (LAURENCON et al. 2003; CIAPPONI et al. 2004; SILVA et al. 2004). mei41^29D rad50^5.1 double mutants were obtained by crossing mei41^29D/FM7-GFP; rad50^5.1/CyO-GFP females to FM7-GFP/Y; rad50^5.1/CyO-GFP males; mei41^29D tefu^atm6 and mei41^29D nbs¹ double mutants were generated by crossing mei41^29D/FM7-GFP; tefu^atm6 (or nbs¹)/TM6B females to FM7-GFP/Y; tefu^atm6 (or nbs¹)/TM6B males; rad50^5.1 nbs¹ double mutants were obtained by inter se mating of rad50^5.1/CyO-GFP; nbs¹/TM6B flies. Chromosomes bearing both nbs¹ and tefu^atm6 were generated by recombination and balanced over TM6C. Doubly mutant larvae from these crosses were unambiguously identified on the basis of their non-GFP and/or non-Tubby phenotypes. The balancers used in these crosses are described in detail in FlyBase (http://flybase.bio.indiana.edu/).

Oregon-R is a standard laboratory wild-type strain. All stocks were maintained, and crosses were made on standard Drosophila medium at 25°.

Chromosome cytology and analysis of telomeric fusions:

DAPI-stained larval brain chromosome preparations were made as described previously (CENCI et al. 1997). For each mutant or doubly mutant brain (Table 1), we examined between 40 and 60 metaphases for the presence of both TAs and chromosome breaks. To assess whether the mean frequencies of TAs detected in the mutants were significantly different, we used the Student's t-test, considering each brain as an independent sample.

View this table: In this window In a new window   TABLE 1 Frequencies of chromosome abnormalities in colchicine-arrested metaphases from mutant larval brains of the indicated genotypes

 

Immunostaining and microscopy:

To obtain preparations for immunostaining, larval brains were dissected in 0.7% sodium chloride, incubated in 0.7% sodium chloride with 10^–5 M colchicine for 45 min, treated with hypotonic solution (0.5% sodium citrate) for 7 min, and then fixed. Fixation and immunostaining for HP1/ORC associated protein (HOAP) and Rad50 were carried out as described by CENCI et al. (2003) and CIAPPONI et al. (2004), respectively.

Polytene chromosome preparations were obtained as described previously (SIRIACO et al. 2002). For double immunostaining of HOAP and HP1, polytene chromosomes were incubated overnight with both the anti-HP1 C1A9 mouse monoclonal and the anti-HOAP rabbit polyclonal antibodies, as described in CIAPPONI et al. (2004).

Mitotic and polytene chromosome preparations were analyzed using a Zeiss Axioplan epifluorescence microscope (Carl Zeiss, Obezkochen, Germany), equipped with a cooled CCD camera (Photometrics, Woburn, MA). Gray-scale digital images were collected separately, converted to Photoshop format, pseudocolored, and merged.

Western blotting:

Twenty brains from third instar larvae of each genotype were dissected in 0.7% NaCl and homogenized in SDS sample buffer, incubated for 5 min at 98°, and subjected to centrifugation for 5 min at 10,000 g. Equal aliquots of the supernatants were separated on 10% SDS–PAGE gels. Western blotting was performed as described previously (CENCI et al. 2003). The anti-Rad50 (CIAPPONI et al. 2004) and anti- tubulin (Sigma, St. Louis) antibodies were used at dilutions of 1:1000 and 1:5000, respectively.

The Drosophila Nbs protein consists of 827 amino acids and displays 23% identity and 38% similarity with its human NBS1 counterpart. Drosophila Nbs contains the three conserved domains present in most Nbs-like proteins: the fork head-associated (FHA) and the BRCA1 C terminus (BRCT) domains at its N terminus and an MRE11-binding domain at the C-terminal region (Figure 1A; IIJIMA et al. 2004). Cytological analysis of brains from larvae homozygous for l(3)67BDp, a lethal mutation mapping close to the nbs gene, revealed the presence of telomeric fusions and chromosome breakage. Sequencing of the nbs gene in the l(3)67BDp mutant strain showed a 238-bp deletion in exon 3, resulting in a truncated Nbs protein of 518 amino acids (Figure 1A). This nbs mutant allele is identical to that recently described by BI et al. (2005) and will be henceforth designated as nbs¹.

View larger version (53K): In this window In a new window Download PPT slide   FIGURE 1.— Mutations in the nbs gene cause telomeric fusion and chromosome breakage and confer extreme sensitivity to X rays. (A) Molecular characterization of the nbs1 mutation. (Top) The wild-type Drosophila Nbs protein that consists of 827 amino acids and contains three conserved domains: the FHA and the BRCT domains and an Mre11-binding domain (Mre11 BD). In the nbs1 mutant allele, a 238-bp deletion causes a frameshift mutation that alters the amino acid sequence from positions 507 to 517 (solid region in the bottom bar) and introduces a stop codon at position 518, resulting in a truncated protein of 517 amino acids. (B) Examples of cytological defects observed in nbs1 mutant brains. (a) Control male metaphase. (b) Mutant female metaphase showing two third chromosomes joined by a DTA (arrow). (c) Mutant female metaphase showing a tricentric configuration consisting of an XR-2 DTA (arrow) and an XL-2 STA (arrowhead). (d) Mutant male metaphase containing an XL-2 DTA (arrow) and a Y-4 DTA (arrowhead). (e) Mutant male metaphase showing three linear dicentric chromosomes generated by a Y-3 DTA (arrowhead), a 2-2 DTA (arrow), and a 4-4 DTA (asterisk). (f) Mutant female metaphase containing a dicentric ring generated by XR-XR and XL-XL DTAs (arrow), a 3-3 DTA (arrowhead), and an isochromatid break involving chromosome 2 (the centric and acentric fragment are indicated by an asterisk and an open arrow, repectively). (C) Frequencies of chromatid and isochromatid breaks observed in nbs1 and tefuatm6 mutants; Oregon-R (wt) was used as a wild-type control. Cells were not exposed to X rays (0 Gy) or irradiated with 1 Gy (n > 100 cells scored for each genotype).

 

Mutations in the nbs gene cause telomeric fusion, chromosome breakage, and apoptosis:

Most individuals homozygous or hemizygous for the nbs¹ mutation die at late larval or pupal stages. In a few cases, we observed mutants dying close to the time of eclosion. These pharate adults consistently showed small and rough eyes (data not shown). This trait is usually associated with high levels of chromosome instability and cell death (AHMAD and GOLIC 1996; BRODSKY et al. 2000) and was previously observed in rad50 mutants (CIAPPONI et al. 2004). Consistent with these results, we also observed that the imaginal discs of nbs¹ mutants were often small and misshapen and displayed apoptotic cells very frequently when stained by acridine orange (data not shown).

To directly assess the role of the nbs gene in the maintenance of chromosome stability, we examined DAPI-stained preparations of colchicine-treated larval brains from nbs¹ mutants. This analysis revealed that homozygous nbs¹ brains display high frequencies of both telomeric associations (TAs) and chromosome breaks (Figure 1B and Table 1 ). TAs were classified as double telomeric associations (DTAs) when they joined a pair of sister telomeres with another pair, and as single telomeric associations (STAs) when they conjoined a single telomere with either its sister telomere or another nonsister telomere. Since DTAs and STAs are thought to be generated during G₁ and S-G₂, respectively (DE LANGE 2002), each type of TA was considered as a single fusion event (Table 1). Telomeric associations observed in nbs¹ mutants involved all chromosome ends (data not shown), as observed in rad50, mre11, UbcD1, Su(var)205, cav, and woc mutants (CENCI et al. 1997, 2003; FANTI et al. 1998; CIAPPONI et al. 2004; RAFFA et al. 2005). Surprisingly, the frequency of TAs observed in nbs¹ mutant brains is approximately one-half of that found in either rad50^5.1 or mre11^DC null mutants (Table 1; CIAPPONI et al. 2004). This is not a consequence of a residual wild-type function of the nbs¹ mutant allele; nbs¹/nbs¹ homozygotes and nbs¹/Df(3L)AC1 hemizygotes display comparable frequencies of TAs, suggesting that the nbs¹ mutation is functionally null (Table 1).

Although nbs¹ mutants have fewer TAs than rad50 and mre11 mutants, the three types of mutants display similar frequencies of chromosome breaks (Table 1; CIAPPONI et al. 2004). These breaks involved either one or both sister chromatids and were characterized by the simultaneous presence of both the acentric and the centric fragment. Thus, they were genuine chromatid and isochromatid breaks and not the consequence of a severed anaphase bridge generated by a TA. To substantiate the finding that mutations in the nbs gene affect mitotic chromosome integrity, we determined the sensitivity of nbs¹ mutants to the induction of chromosome breakage by X rays. Since the Drosophila ATM kinase encoded by the tefu gene is thought to be in the same telomere protection pathway with the Mre11/Rad50 complex (BI et al. 2004), we also examined the radiosensitivity of tefu^atm6 mutants; previous studies have shown that tefu^atm6 is a functionally null allele (SILVA et al. 2004). This analysis showed that nbs¹ mutants irradiated with 1 Gy of X rays exhibit 10-fold more chromosome breaks than the Oregon-R control irradiated with the same dose (Figure 1C). Similar results were previously obtained by irradiating mre11 and rad50 mutants (CIAPPONI et al. 2004). However, the X-ray sensitivity of tefu^atm6 mutants was substantially lower, as they displayed only 4-fold more chromosome breaks than the Oregon-R controls (Figure 1C).

Nbs is required for HP1/HOAP localization at telomeres:

To define the role of the nbs gene in telomere protection, we asked whether it is required for proper localization of HP1 and HOAP at chromosome ends. HP1 and HOAP form a complex that protects Drosophila telomeres from fusion events (reviewed by CENCI et al. 2005). Because HP1 cannot be readily detected at mitotic chromosome ends (FANTI et al. 1998), we examined HOAP localization in both mitotic and polytene chromosomes and HP1 localization only in polytene chromosomes. The analysis of mitotic chromosomes showed that mutations in the nbs gene affect HOAP localization at telomeres. In nbs¹ mutants, 45% (n = 247) of the telomeres not involved in fusions displayed a clear HOAP signal, whereas in the Oregon-R control the frequency of labeled telomeres was 89% (n = 158; Figure 2A). Consistent with these results, only 25% (n = 100) of the polytene chromosome telomeres from nbs¹ mutants showed low but detectable HOAP and HP1 accumulations, while the remaining 75% of mutant telomeres did not exhibit any HP1/HOAP signal (Figure 2B). In contrast, 96% (n = 100) of the Oregon-R polytene chromosome ends displayed strong HOAP/HP1 signals. These results differ from those obtained with rad50 and mre11 mutants. In mre11 and rad50 mutants, the frequencies of mitotic telomeres with detectable HOAP signals were 5.5 and 18.2%, respectively, and the polytene chromosome telomeres were always devoid of HP1/HOAP signals (CIAPPONI et al. 2004). Thus, mutations in the nbs gene have milder effects on HP1/HOAP accumulation at telomeres than mutations in either mre11 or rad50, consistent with the relatively low frequency of TAs observed in nbs mutants.

View larger version (21K): In this window In a new window Download PPT slide   FIGURE 2.— Nbs is required for proper telomeric localization of both HOAP and HP1. (A) HOAP localization (red) on mitotic chromosomes from Oregon-R (wt) and nbs1 larval brain cells. The nbs1 mutant cell displays a 3-4 DTA (arrow). (B) Simultaneous HOAP (red) and HP1 (green) immunolocalization on the distal segment of the XL arm from wild-type (wt) and nbs1 (two examples) polytene chromosomes. In nbs1 mutants, accumulation of both HP1 and HOAP at telomeres is either completely absent (middle) or strongly reduced (right). Arrows point to the telomeres.

 

Nbs is required for Rad50 localization in interphase nuclei:

Previous work has shown that in Drosophila mre11 mutants the rad50 gene is normally transcribed but the Rad50 protein is not detectable by Western blotting, suggesting that Rad50 is unstable in the absence of its binding partner Mre11 (CIAPPONI et al. 2004). These results are consistent with studies in human cells showing that in MRE11 mutant cells there is no detectable expression of RAD50 and the expression of NBS1 is reduced (STEWART et al. 1999; UZIEL et al. 2003; STRACKER et al. 2004). However, mammalian NBS1 is not essential for the stability of the MRE11 and RAD50 proteins but is required to transport (or retain) these proteins in the nucleus (CARNEY et al. 1998; MASER et al. 2001; DIFILIPPANTONIO et al. 2005). We thus asked whether Drosophila Nbs is required for the stability and nuclear localization of its binding partner Rad50. Because Drosophila ATM is also involved in telomere protection, we also asked whether this kinase is required for normal behavior of Rad50. As shown in Figure 3A, mutations in the nbs¹ and tefu^atm6 genes do not affect the stability of the Rad50 protein. However, examination of nbs¹ mutant brains immunostained for Rad50 revealed that the Nbs protein is required for nuclear localization of Rad50. Whereas in wild-type brains Rad50 is concentrated in the nucleus, in nbs¹ mutant brains Rad50 accumulates is the cytoplasm (Figure 3B). Nonetheless, in 42% (n = 250) of the prometaphase/metaphase figures from nbs¹ mutant brains, Rad50 is associated with the chromosomes (Figure 3C); in wild-type brains, 63% (n = 330) of the mitotic figures are immunostained by the anti-Rad50 antibody (Figure 3C). These results suggest that, in late G₂, part of the cytoplasmic pool of Rad50 can enter the nucleus in the absence of Nbs. In tefu^atm6 mutants, Rad50 is regularly enriched at both the interphase nuclei and the chromosomes; 60% of prometaphase/metaphase figures from tefu^atm6 brains are immunostained by the anti-Rad50 antibody (Figure 3B).

View larger version (33K): In this window In a new window Download PPT slide   FIGURE 3.— Mutations in the nbs gene do not affect Rad50 expression but prevent its nuclear localization. (A) Immunoblot from Oregon-R (wt), nbs1, tefuatm6, rad505.1, and mre11DC larval brain homogenates. Note the absence of the Rad50 protein in both the rad50 and the mre11 mutants. In contrast, the Rad50 expression levels in nbs1 and tefuatm6 mutants are comparable to wild type. -Tubulin was used as a loading control. (B) Localization of the Rad50 protein in interphase cells from Oregon-R (wt), nbs1, and tefuatm6 brains. Cells were stained with DAPI (green) and with the anti-Rad50 antibody (red). Note that the Rad50 protein colocalizes with nuclear DNA in both control and tefuatm6 mutant cells; in the nbs1 mutant cells, Rad50 is excluded from the nucleus and remains in the cytoplasm. (C) Localization of the Rad50 protein in colchicine-arrested metaphase chromosomes from Oregon-R (wt), nbs1, and tefuatm6 larval brains. Metaphase preparations were stained with DAPI and with the anti-Rad50 antibody. The arrows in atm6 and nbs1 point to an autosomal monocentric ring generated by a DTA and a XR-XR DTA, respectively.

 

Functional relationships among the nbs, rad50, tefu, and mei-41 genes:

The partial exclusion of Rad50 protein from the nucleus of nbs brain cells raises the possibility that the telomere fusion phenotype observed in nbs mutants is simply the consequence of a reduced intranuclear concentration of Rad50. However, it is also possible that Nbs plays additional and more direct roles in Drosophila telomere protection. To obtain insight into the telomere-related functions of the Nbs protein, we analyzed the interaction between the nbs¹ mutation and mutations in other genes known to be involved in telomere protection. Previous studies have shown that the frequencies of TAs observed in rad50 mre11 and mre11 tefu double mutants are approximately the same as those found in flies homozygous for the single mutations (BI et al. 2004; CIAPPONI et al. 2004). These results indicate that mre11, rad50, and atm are involved in a single pathway of Drosophila telomere protection. In contrast, epistasis analysis with mei-41 (that encodes an ATR homolog) showed that both mei-41 mre11 and mei-41 tefu double mutants exhibit frequencies of TAs significantly higher than those observed in strains bearing the single mutations (BI et al. 2005). Similar results were obtained when the epistasis analysis was performed with mus-304, the Drosophila gene that encodes the ATR-interacting protein ATRIP (BRODSKY et al. 2000); mus-304 mre11 and mus-304 tefu double mutants showed more TAs than the single mutants (BI et al. 2005). These findings indicate that the Drosophila telomere protection pathway identified by the Rad50/Mre11 complex and the ATM kinase is different from the pathway specified by the ATR/ATRIP complex. Moreover, since mei-41 and mus-304 mutant flies do not exhibit telomere fusions, it has been suggested that the ATR-controlled pathway is redundant (BI et al. 2005).

To analyze the epistasis relationships of the nbs¹ mutation, we constructed rad50^5.1 tefu^atm6, mei-41^29D nbs¹, rad50^5.1 nbs¹, and nbs¹ tefu^atm6 double mutants and compared their phenotypes with those of the corresponding single mutants (Table 1); the mei-41^29D mutant allele is functionally null (LAURENCON et al. 2003), like the rad50^5.1, nbs¹, and tefu^atm6 mutations (CIAPPONI et al. 2004; SILVA et al. 2004; this work). The results of this analysis showed that the rad50^5.1 tefu^atm6 double mutant has a frequency of TAs comparable to those observed in the single mutants, consistent with the finding that mre11 and tefu function in the same pathway of telomere protection (BI et al. 2005). In contrast, the mei-41^29D nbs¹ double mutant displayed a higher frequency of TAs than either single mutant (Table 1), in agreement with previous findings of BI et al. (2005). However, we also found that the levels of TAs in nbs¹ tefu^atm6 and rad50^5.1 nbs¹ double mutants are significantly higher than those observed in the corresponding single mutants (Figure 1B and Table 1). These results indicate that nbs, rad50, and tefu do not function in a single pathway for the protection of Drosophila telomeres.

Our cytological analyses showed that the rad50^5.1 and nbs¹ single mutants and the nbs¹ rad50^5.1 double mutant all exhibit comparable frequencies of chromosome breaks (Table 1). Thus, although Nbs and Mre11/Rad50 make distinct contributions to telomere protection, the three components of the MRN complex appear to act in a single pathway to prevent spontaneous chromosome breakage. However, nbs¹ tefu^atm6, rad50^5.1 tefu^atm6, mei-41^29D nbs¹, and mei-41^29D rad50^5.1 double mutants displayed frequencies of chromosome breaks significantly higher than those observed in the corresponding single mutants (Table 1). These results indicate that the MRN complex and the kinases ATM and ATR function in different pathways to prevent chromosome breakage.

We have shown that the wild-type function of the Drosophila nbs gene is required to maintain chromosome integrity and to prevent telomere fusion. Our results indicate that the nbs, mre11, and rad50 genes function in single pathway for the repair of spontaneous DNA lesions leading to chromosome breakage. In addition, we have found that double mutants affecting a single component of the MRN complex and either the ATM or the ATR kinase exhibit more chromosome breaks than the corresponding single mutants. The simplest interpretation of these results is that the two kinases function in multiple pathways for the repair of the DNA damage leading to chromosome breakage and that some of these pathways do not include the MRN complex. The finding that mei-41^29D tefu^atm6 double mutants and tefu^atm6 single mutants display similar frequencies of chromosome breaks (Table 1) indicates that the ATM and ATR play redundant roles in the protection from spontaneous chromosome breakage. However, tefu and mei-41 mutants are four- and eightfold more sensitive than wild type to the X-ray induction of chromosome breakage, respectively (this work; GATTI et al. 1980). Thus, ATR may play a principal role in the repair of the lesions leading to chromosome breaks, with ATM playing a backup role.

Recent work has shown that ATM and ATR/ATRIP function in different but redundant pathways of Drosophila telomere protection, with ATM playing an essential role and ATR compensating for the loss of ATM activity (BI et al. 2005). Here, we have shown that the frequencies of TAs observed in nbs tefu and rad50 nbs double mutants are significantly higher than those observed in the corresponding single mutants (Table 1). An interpretation of these findings is that the Nbs protein functions in a telomere protection pathway that is different from either the ATR/ATRIP or the ATM/Rad50/Mre11 pathway. Alternatively, Nbs could function in both the ATM- and ATR-controlled pathways. These results are at odds with those obtained in budding yeast, where Tel1 (the ATM homolog), Rad50, Mre11, and Xrs2 (the NBS homolog) function in a single pathway of telomere maintenance (RITCHIE and PETES 2000). However, they are consistent with several results obtained in human cells, showing that the NBS and the MRE11/RAD50 components of the MRN complex can function independently. For example, it has been shown that NBS1 and the MRE11/RAD50 complex have separate roles in both ATM activation and ATM-mediated phosphorylation events (CEROSALETTI and CONCANNON 2004; LEE and PAULL 2004). Moreover, while NBS1 localization to the human telomeres is restricted to the S phase, the MRE11/RAD50 complex remains associated with telomeres throughout the cell cycle (ZHU et al. 2000).

Our results suggest a model for the role of Nbs in Drosophila telomere protection. This model is based on the assumption that Nbs can facilitate both ATR- and ATM-mediated phosphorylation events, as recently shown in mammalian systems (STIFF et al. 2005). We propose that Nbs is involved in both the Rad50/Mre11/ATM and the ATR/ATRIP telomere protection pathways. Nbs would mediate the transport of the Rad50/Mre11 complex in the nucleus in the Rad50/Mre11/ATM pathway and facilitate certain ATR-mediated phosphorylation events in the ATR/ATRIP pathway (Figure 4). Taking into account that the ATR/ATRIP telomere protection pathway is redundant (BI et al. 2005), our model can explain the results of the epistasis analysis. We speculate that in nbs mutants both pathways are partially impaired, resulting in a relatively low frequency of TAs (Table 1). In rad50 nbs and tefu nbs double mutants, the Rad50/Mre11/ATM pathway would be disrupted and the ATR/ATRIP pathway partially impaired, resulting in TA frequencies higher than those found in the single mutants (Table 1). Finally, in mei-41 tefu, mus-304 tefu, mei-41 rad50, and mei-41 mre11 double mutants, both pathways would be disrupted, resulting in very high frequencies of TAs (BI et al. 2005).

View larger version (34K): In this window In a new window Download PPT slide   FIGURE 4.— A model illustrating the possible roles of the Nbs protein in Drosophila telomere protection. We propose that Nbs mediates transport of Mre11/Rad50 in the nucleus and facilitates ATR-mediated phosphorylation of a critical telomere-associated target. The precise relationships between the Mre11/Rad50 complex and ATM are unclear, as the MRN complex is required for both ATM activation and ATM-mediated phosphorylation events. It is also unknown whether ATM-mediated phosphorylation of Nbs and Mre11 (thin arrows) plays a role in telomere protection. See text for further explanation.

 
An aspect of the phenotype that is difficult to explain is the pattern of HOAP localization in different mutants and double mutants. In the mre11 and rad50 mutants, most mitotic telomeres are devoid of the HOAP protein (BI et al. 2004; CIAPPONI et al. 2004). In nbs mutants, the frequency of telomeres with detectable HOAP accumulations is lower than in wild type but higher than in either the mre11 or the rad50 mutant, consistent with a reduced intranuclear concentration of the Rad50/Mre11 complex. tefu (ATM) and mei-41 (ATR) single mutants have normal HOAP concentrations at mitotic telomeres (BI et al. 2004) but in mei-41 tefu double mutants telomeres lack the HOAP protein (BI et al. 2005). Normal HOAP accumulations at mitotic telomeres were also found in Su(var)205 (HP1) and woc mutants that display very high frequencies of TAs, indicating that the presence of HOAP at chromosome ends is not sufficient to ensure proper telomere protection (CENCI et al. 2003; RAFFA et al. 2005). An interpretation of these results is rather difficult, mainly because our current knowledge of the Drosophila telomere components is largely incomplete. HOAP localization at telomeres may be mediated, not only by the Rad50/Mre11 complex, but also by a factor that needs to be phosphorylated by both ATM and ATR. When this factor is not phosphorylated at its ATM-dependent site(s), telomeres are deprotected even if they accumulate normal amounts of HOAP. However, when this factor is not phosphorylated in both its ATM- and ATR-dependent sites, telomeres lose their ability to recruit HOAP. This factor cannot be HOAP itself, as recent work has shown that the HOAP protein is not phosphorylated in a wild-type background (BI et al. 2005).

We have shown that the Drosophila Nbs protein is required for transport of Rad50 in the nucleus and for prevention of telomere fusion and chromosome breakage. In addition, our results indicate that Nbs can act independently of the Rad50/Mre11 complex. Remarkably, all these features of the Drosophila Nbs protein are shared by its human counterpart (CARNEY et al. 1998; MASER et al. 2001; CEROSALETTI and CONCANNON 2004; LEE and PAULL 2004; DIFILIPPANTONIO et al. 2005; ZHANG et al. 2005). The ATLD disorder caused by hipomorphic mutations in the MRE11 gene and NBS have many overlapping features but are clinically distinct. NBS patients are characterized by microcephaly and developmental delay, while ATLD patients exhibit a mild ataxia telangiectasia-like phenotype with no microcephaly and no developmental delay (reviewed by STRACKER et al. 2004). Given the functional similarities within Drosophila and human NBS proteins, it is likely that further studies on the Drosophila MRN complex will help to elucidate the molecular basis of the clinical differences between ATLD and NBS.

We are particularly grateful to Byron Williams and Mike Golberg for telling us that l(3)DBp67 mutants display telomeric fusions. We thank Rebecca Kellum and Sally Elgin for the anti-HOAP and anti-HP1 antibodies and Shelag Campbell and Tin Tin Su for the tefu^atm6 and mei41^29D mutant alleles. L.C. was supported by the "Rientro cervelli" program by Ministero dell'Università e della Ricerca. This work has been supported in part by grants from Italian Telethon and from the Associazione Italiana per la Ricerca sul Cancro to M.G.

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