Nijmegen breakage syndrome: Biological Overview | Developmental Biology | Effects of Mutation | Evolutionary Homologs | References
Gene name - Nijmegen breakage syndrome
Cytological map position- 67C5-67C5
Function - signaling
Symbol - nbs
FlyBase ID: FBgn0261530
Genetic map position - 3L
Classification - fork head-associated (FHA) domain, BRCA1 C terminus (BRCT) domain
Cellular location - nuclear and cytoplasmic
Two protein kinases ATM and ATR as well as the Mre11/Rad50/Nbs (MRN) complex, which contains two highly conserved proteins Mre11 and Rad50 and a third less-conserved component, Nbs/Xrs2 (also known as nibrin), play critical roles in the response to DNA damage and telomere maintenance in mammalian systems. The primary function of the MRN complex is to sense DNA strand breaks and then to amplify the initial signal and convey it to downstream effectors, such as ATM, p53, Nbs1 (as a target of ATM), SMC1 and Brca1, that regulate cell cycle checkpoints and DNA repair. Mre11-Rad50 can bind DNA and that Mre11 possesses a nuclease activity that can process these ends. Nbs stimulates the DNA binding and nuclease activity by Mre11-Rad50. In vivo, Nbs is responsible for translocating the MRN complex to the nucleus and relocalizing the complex to the sites of DSBs following irradiation. The MRN complex is also required for activation of the S-phase checkpoint following DNA damage (Ciapponi, 2006).
It has been shown that mutations in the Drosophila mre11 and rad50 genes cause both telomere fusion and chromosome breakage. This study 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 (Ciapponi, 2006).
The MRN complex plays critical roles in the response to DNA damage and telomere maintenance in both yeast and mammalian systems (D'Adda Di Fagagna, 2004; Stracker, 2004; Zhang, 2005; Lee, 2006). 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, 2004; Stracker, 2004). These clinical differences are likely to reflect functional differences between the Nbs and Mre11 components of the human MRN complex (Ciapponi, 2006).
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, 2003; J.-H. Lee, 2004, 2005; Cerosaletti, 2006). The MRN complex also enhances several ATR-dependent phosphorylation events (Stiff, 2005; Zhong, 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, 2004; Lee, 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 (Ciapponi, 2006 and references therein).
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. 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. 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. This study explored the role of the Drosophila nbs gene in both telomere protection and the maintenance of chromosome integrity. The 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 (Ciapponi, 2006).
This study shows that the wild-type function of the Drosophila nbs gene is required to maintain chromosome integrity and to prevent telomere fusion. The 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, it was 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-4129D tefuatm6 double mutants and tefuatm6 single mutants display similar frequencies of chromosome breaks 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. Thus, ATR may play a principal role in the repair of the lesions leading to chromosome breaks, with ATM playing a backup role (Ciapponi, 2006).
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, 2005). This study shows 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. 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, 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, 2004; Lee, 2004). Moreover, while NBS1 localization to the human telomeres is restricted to the S phase, the MRE11/RAD50 complex (Zhu, 2000) remains associated with telomeres throughout the cell cycle (Ciapponi, 2006).
The 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, 2005). It is proposed 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. Taking into account that the ATR/ATRIP telomere protection pathway is redundant (Bi, 2005), the model can explain the results of the epistasis analysis. It is speculated that in nbs mutants both pathways are partially impaired, resulting in a relatively low frequency of TAs. 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. 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, 2005) (Ciapponi, 2006).
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. 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, 2004) but in mei-41 tefu double mutants telomeres lack the HOAP protein (Bi, 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. An interpretation of these results is rather difficult, mainly because the 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 (Bi, 2005) has shown that the HOAP protein is not phosphorylated in a wild-type background (Ciapponi, 2006).
This study has 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, the 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, 1998; Maser, 2001; Cerosaletti, 2004; Lee, 2004; Difilippantonio, 2005; Zhang, 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, 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 (Ciapponi, 2006).
In higher eukaryotes, the ataxia telangiectasia mutated (ATM) and ATM and Rad3-related (ATR) checkpoint kinases play distinct, but partially overlapping, roles in DNA damage response. Yet their interrelated function has not been defined for telomere maintenance. The two proteins control partially redundant pathways for telomere protection in Drosophila: the loss of ATM (encoded by telomere fusion) leads to the fusion of some telomeres, whereas the loss of both ATM and ATR (encoded by mei-41) renders all telomeres susceptible to fusion. The ATM-controlled pathway includes the Mre11 and Nijmegen breakage syndrome complex but not the Chk2 kinase, whereas the ATR-regulated pathway includes its partner ATR-interacting protein but not the Chk1 kinase. This finding suggests that ATM and ATR regulate different molecular events at the telomeres compared with the sites of DNA damage. This compensatory relationship between ATM and ATR is remarkably similar to that observed in yeast despite the fact that the biochemistry of telomere elongation is completely different in the two model systems. Evidence is provided suggesting that both the loading of telomere capping proteins and normal telomeric silencing require ATM and ATR in Drosophila and it is proposed that ATM and ATR protect telomere integrity by safeguarding chromatin architecture that favors the loading of telomere-elongating, capping, and silencing proteins (Bi, 2005).
This study defines two partially redundant pathways, regulated by ATM and ATR, respectively, that ensure complete protection of Drosophila telomeres. It is further suggested that two other proteins, Meiotic recombination 11 (Mre11) and Nbs belong to the same ATM-regulated pathway, whereas ATR-Interacting Protein (ATRIP or Mutagen-sensitive 304) participates in the ATR-controlled pathway. This conclusion would be consistent with the pathway components defined in yeasts. Based on the facts that Drosophila tefu mutants have widespread telomere fusions that eventually lead to lethality, whereas mei-41 mutants are viable with no apparent telomeric defects, it is proposed that ATM is more important in regulating telomere protection, with ATR playing a backup role. In mei-41 mutants, ATM may fully compensate ATR's absence on telomeres to ensure complete capping throughout the cell cycle. In contrast, ATR in tefu mutants may partially compensate for the loss of ATM because it is a less efficient regulator of capping as suggested earlier. This partial redundancy leads to the fusion of some telomeres (Bi, 2005).
ATM, ATR, and their cofactors are known to control multiple checkpoints in response to DNA damage and abnormal telomeres. Perhaps cells with uncapped telomeres are allowed to continue cycling because of defective checkpoints and that leads to the fusion of these telomeres. This possibility is considered unlikely based on several observations. (1) In cav1 mutant (see caravaggio), telomere fusion occurs at a high rate even in the presence of a full complement of checkpoint genes. (2) No exacerbated telomere dysfunction is found in either the cav1 tefu (atm) or the cav1 mei-41 (atr) double mutant. (3) Mutations in Drosophila chk1 or chk2 did not affect existing telomere defects in either tefu or mre11 mutant, suggesting that checkpoints jointly controlled by these effectors and the respective upstream kinases do not normally respond to dysfunctional telomeres. Therefore, the contribution to the telomere dysfunction from checkpoint defects is likely small in the cases studied (Bi, 2005).
Cytological analyses suggest that one of the functions of ATM and ATR at Drosophila telomeres is to facilitate the loading of telomere capping and silencing proteins, such as HOAP (Caravaggio) and HP1, and they do so in a partially redundant fashion. In the case of HOAP's binding to mitotic telomeres, either ATM or ATR is sufficient for its normal loading. When both kinases are absent, HOAP can no longer be detected at the telomeres. It was interesting that there were more telomere fusions in either tefu mei-41D9 or tefu mus304D1/D3 than in either mre11 or nbs1 cells, yet normal HOAP signals were detected on fusion-free telomeres for only the first two genotypes. It is known that the presence of HOAP at chromosome ends is not sufficient to prevent fusion, which suggests that the loss of other capping proteins could also lead to fusion. That may be the case in tefu mei-41D9 and tefu mus304D1/D3 cells. It is also known that the absence of HOAP at any particular telomere does not necessarily lead to fusion, suggesting other capping proteins can sometimes compensate for HOAP's function. That idea, in contrast, may apply to the situation in cells deficient for the Mre11 complex (Bi, 2005).
The mechanism by which ATM and ATR maintain telomere integrity remains largely unclear. Because the conserved kinase domain of both ATM and ATR is essential for normal telomere function in S. cerevisiae, ATM and ATR likely exert their function by protein phosphorylation. Neither Chk1 nor Chk2 was involved in telomere protection in Drosophila, a situation similar to S. pombe, which suggests that ATM and ATR modify a different set of proteins at telomeres. ATM and ATR may regulate HOAP's capping activity by directly phosphorylating HOAP. However, no evidence was recovered that HOAP is phosphorylated in WT cells. Therefore, ATM and ATR may indirectly regulate HOAP's ability to bind telomeres, perhaps by modulating telomeric structure. The results support the hypothesis that ATM and ATR have a conserved function at the telomeres that is independent of telomerase. Because Drosophila telomeres are not elongated by a telomerase, the fly may be an excellent system for studying the roles of ATM and ATR in telomere protection, uncoupled from their roles in telomere elongation (Bi, 2005).
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, mutants were observed dying close to the time of eclosion. These pharate adults consistently showed small and rough eyes. This trait is usually associated with high levels of chromosome instability and cell death and was previously observed in rad50 mutants (Ciapponi, 2004). Consistent with these results, it was 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 (Ciapponi, 2006).
To directly assess the role of the nbs gene in the maintenance of chromosome stability, DAPI-stained preparations of colchicine-treated larval brains were examined from nbs1 mutants. This analysis revealed that homozygous nbs1 brains display high frequencies of both telomeric associations (TAs) and chromosome breaks. 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, each type of TA was considered as a single fusion event. Telomeric associations observed in nbs1 mutants involved all chromosome ends, as observed in rad50, mre11, UbcD1, Su(var)205, cav, and woc mutants. Surprisingly, the frequency of TAs observed in nbs1 mutant brains is approximately one-half of that found in either rad50Δ5.1 or mre11DC null mutants. 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 (Ciapponi, 2006).
Although nbs1 mutants have fewer TAs than rad50 and mre11 mutants, the three types of mutants display similar frequencies of chromosome breaks. 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, the sensitivity of nbs1 mutants to the induction of chromosome breakage by X rays was determined. 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, 2004), the radiosensitivity of tefuatm6 mutants was examined; previous studies have shown that tefuatm6 is a functionally null allele. 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. Similar results were previously obtained by irradiating mre11 and rad50 mutants. However, the X-ray sensitivity of tefuatm6 mutants was substantially lower; they displayed only 4-fold more chromosome breaks than the Oregon-R controls (Ciapponi, 2006).
Nbs is required for HP1/HOAP localization at telomeres: To define the role of the nbs gene in telomere protection, it was asked whether nbs 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, 2005). Because HP1 cannot be readily detected at mitotic chromosome ends, HOAP localization was examined in both mitotic and polytene chromosomes and HP1 localization was examined 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% 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%. Consistent with these results, only 25% 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. In contrast, 96% 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, 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 (Ciapponi, 2006).
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, 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, 1999; Uziel, 2003; Stracker, 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, 1998; Maser, 2001; Difilippantonio, 2005). It was 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, it was also asked whether this kinase is required for normal behavior of Rad50. Mutations in the nbs1 and tefuatm6 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. Nonetheless, in 42% of the prometaphase/metaphase figures from nbs1 mutant brains, Rad50 is associated with the chromosomes; in wild-type brains, 63% of the mitotic figures are immunostained by the anti-Rad50 antibody. These results suggest that, in late G2, part of the cytoplasmic pool of Rad50 can enter the nucleus in the absence of Nbs. In tefuatm6 mutants, Rad50 is regularly enriched at both the interphase nuclei and the chromosomes; 60% of prometaphase/metaphase figures from tefuatm6 brains are immunostained by the anti-Rad50 antibody (Ciapponi, 2006).
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, the interaction between the nbs1 mutation and mutations in other genes known to be involved in telomere protection were analyzed. 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, 2004; Ciapponi, 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, 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, 2000); mus-304 mre11 and mus-304 tefu double mutants showed more TAs than the single mutants (Bi, 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 (Bi, 2005) that the ATR-controlled pathway is redundant (Ciapponi, 2006).
To analyze the epistasis relationships of the nbs1 mutation, rad50Δ5.1 tefuatm6, mei-4129D nbs1, rad50Δ5.1 nbs1, and nbs1 tefuatm6 double mutants were constructed and their phenotypes were compared with those of the corresponding single mutants; the mei-4129D mutant allele is functionally null, like the rad50Δ5.1, nbs1, and tefuatm6 mutations. The results of this analysis showed that the rad50Δ5.1 tefuatm6 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, 2005). In contrast, the mei-4129D nbs1 double mutant displayed a higher frequency of TAs than either single mutant, in agreement with previous findings of Bi (2005). However, it was also found that the levels of TAs in nbs1 tefuatm6 and rad50Δ5.1 nbs1 double mutants are significantly higher than those observed in the corresponding single mutants. These results indicate that nbs, rad50, and tefu do not function in a single pathway for the protection of Drosophila telomeres (Ciapponi, 2006).
Cytological analyses showed that the rad50Δ5.1 and nbs1 single mutants and the nbs1 rad50Δ5.1 double mutant all exhibit comparable frequencies of chromosome breaks. 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 tefuatm6, rad50Δ5.1 tefuatm6, mei-4129D nbs1, and mei-4129D rad50Δ5.1 double mutants displayed frequencies of chromosome breaks significantly higher than those observed in the corresponding single mutants. These results indicate that the MRN complex and the kinases ATM and ATR function in different pathways to prevent chromosome breakage (Ciapponi, 2006).
Analysis of terminal deletion chromosomes indicates that a sequence-independent mechanism regulates protection of Drosophila telomeres. Mutations in Drosophila DNA damage response genes such as atm/tefu, mre11, or rad50 disrupt telomere protection and localization of the telomere-associated proteins HP1 and HOAP, suggesting that recognition of chromosome ends contributes to telomere protection. However, the partial telomere protection phenotype of these mutations limits the ability to test if they act in the epigenetic telomere protection mechanism. The roles were examined of the Drosophila atm and atr-atrip DNA damage response pathways and the nbs homolog in DNA damage responses and telomere protection. As in other organisms, the atm and atr-atrip pathways act in parallel to promote telomere protection. Cells lacking both pathways exhibit severe defects in telomere protection and fail to localize the protection protein HOAP to telomeres. Drosophila nbs is required for both atm- and atr-dependent DNA damage responses and acts in these pathways during DNA repair. The telomere fusion phenotype of nbs is consistent with defects in each of these activities. Cells defective in both the atm and atr pathways were used to examine if DNA damage response pathways regulate telomere protection without affecting telomere specific sequences. In these cells, chromosome fusion sites retain telomere-specific sequences, demonstrating that loss of these sequences is not responsible for loss of protection. Furthermore, terminally deleted chromosomes also fuse in these cells, directly implicating DNA damage response pathways in the epigenetic protection of telomeres. It is proposed that recognition of chromosome ends and recruitment of HP1 and HOAP by DNA damage response proteins is essential for the epigenetic protection of Drosophila telomeres. Given the conserved roles of DNA damage response proteins in telomere function, related mechanisms may act at the telomeres of other organisms (Oikemus, 2006).
The ends of eukaryotic chromosomes can be protected from end-to-end fusion by two distinct mechanisms. In most organisms, sequence-specific DNA binding proteins recognize telomere-specific sequences and protect telomeres from the activity of DNA repair systems. However, genetic studies in Drosophila have demonstrated that telomeres can also be protected from end-to-end fusion by an epigenetic mechanism. The telomeric DNA of Drosophila chromosomes is composed of retrotransposons and repetitive telomere-associated sequences. Terminal deletion chromosomes that completely lack these sequences can be recovered and propagated. The telomeres of these chromosomes are protected from fusion and do not induce DNA damage responses such as cell cycle arrest or apoptosis. These observations demonstrate that a sequence-independent mechanism can protect Drosophila chromosomes from telomere fusion and suggest that a similar mechanism contributes to protection of normal telomeres. The sequence-independent inheritance of telomere protection is conceptually similar to the epigenetic regulation of centromere function in which the function of a chromosomal domain is usually associated with a specific set of sequences, but can be stably transferred to alternative sequences. Thus, Drosophila telomere protection can be grouped with centromere function and gene expression as processes that can be regulated by an epigenetic mechanism (Oikemus, 2006 and references therein).
Two chromatin-associated proteins, HP1 and HOAP, are required for telomere protection and localize to the telomeres of both normal and terminally deleted chromosomes. The role of HP1 in the epigenetic inheritance of chromatin modifications during cell division suggests that a similar activity may contribute to telomere protection. Inheritance of chromatin modifications is often initiated or stabilized by specific chromosome features, such as binding sites for sequence-specific DNA binding proteins or repeat sequences at centromeres. The stable inheritance of terminally deleted chromosomes over many generations indicates that a feature of telomeres other than telomere-specific sequences can recruit or maintain HP1 and HOAP at telomeres (Oikemus, 2006 and references therein).
One signature of telomeres that might contribute to HP1 and HOAP recruitment is the chromosome end itself. Studies in yeast and mammalian cells have demonstrated that telomere protection requires proteins that act at broken chromosome ends during the cellular response to DNA damage; these include the ATM and ATR protein kinases and the Mre11/Rad50/NBS1 (MRN) DNA repair complex. Analysis of cells lacking telomerase and ATM suggests that ATM plays a particularly critical role in cells with short telomeres. Such cells may be least able to utilize sequence-specific mechanisms for telomere protection. In both budding and fission yeast, the combined loss of the ATM and ATR pathways results in severe telomere protection defects. In mammalian cell culture, acute inhibition of the MRN complex or of the ATM and ATR kinases also induces telomere fusions. Drosophila homologs of most DNA damage response genes have been described. The Drosophila telomere fusion (tefu) gene is required to prevent fusions in proliferating cells and is encoded by the Drosophila homolog of ATM. Mutations in the Drosophila DNA damage response genes tefu, mre11, and rad50 lead to partial loss of telomere protection and reduced recruitment of HP1 and HOAP to telomeres. Thus, a DNA damage response pathway contributes to the protection of Drosophila telomeres; however, HP1 and HOAP can also mediate some degree of telomere protection in the absence of this pathway (Oikemus, 2006 and references therein).
Characterized here are the role of Nbs and the ATM and ATR DNA damage response pathways in the epigenetic protection of Drosophila telomeres. In humans, mutations in Nbs1 or ATM result in similar inherited syndromes. In both mammals and yeast, Nbs1 forms a complex with Mre11 and Rad50 (the MRN complex) that acts in the ATM pathway in response to DNA damage and is required for DNA repair and telomere function. This study demonstrates that Drosophila nbs is required for atm- and atr-dependent DNA damage responses including DNA repair. Drosophila mei-41 (the ATR homolog) and mus304 (the ATRIP homolog) act in parallel to the atm pathway in telomere protection; cells lacking both pathways fail to recruit HOAP to the telomeres of mitotic chromosomes and exhibit a severe telomere fusion phenotype. The telomere fusion defect in nbs mutants suggests that it acts in both the tefu and mei-41-mus304 telomere protection pathways and in the chromosome joining step. Advantage was taken of the severe telomere fusion phenotype in cells lacking both pathways to test the role of DNA damage response pathways in the sequence-independent protection of Drosophila telomeres. Analysis of these cells reveals that loss of telomeric HOAP and telomere fusions are not due to loss of telomeric sequences. Furthermore, these DNA damage response pathways are also required to protect the telomeres of terminally deleted chromosomes, directly demonstrating that the DNA damage response pathways are required for epigenetic regulation of telomere protection (Oikemus, 2006).
Drosophila Nbs is required for normal development: To identify genes that cooperate with atm/tefu in telomere protection, mutations were characterized in other Drosophila DNA damage response genes including nbs. Drosophila nbs encodes a protein with N-terminal FHA and BRCT domains and a short region of similarity to the Mre11 interaction domain encoded by human Nbs1. To identify mutations in Drosophila nbs, a collection of lethal mutations in the genetic region containing nbs were screened for pupal lethality and excess apoptosis during wing development, phenotypes previously described for Drosophila tefu, mre11, and rad50. Two mutations with these phenotypes failed to complement each other and their lethality was rescued by a transgene containing the nbs genomic region. Sequencing of these mutations revealed that l(3)67BDp1 (nbs1) contains a 238-base pair (bp) deletion and 1bp insertion that disrupts the open reading frame while l(3)67BDr1 (nbs2 ) introduces a stop codon that truncates the reading frame at amino acid position 685. Both of these mutations are predicted to eliminate the ability of Nbs to interact with Mre11 (Oikemus, 2006).
Flies homozygous for the nbs1 mutation die as pharate adults with rough eyes and missing or abnormal bristles. In tefu, mre11, or rad50 mutant flies, this phenotype is accompanied by increased genomic instability and apoptosis. tefu, but not mei-41 or mus304, is also required for rapid induction (within 4 h) of additional apoptosis by X-irradiation. The developing wings of nbs mutant animals also exhibit high levels of spontaneous apoptosis compared to wild-type animals. X-irradiation of these discs does not induce the rapid, large increase in apoptosis observed in wild-type discs. These results suggest that nbs acts in the tefu DNA damage response pathway to regulate apoptosis. Consistent with this conclusion, nbs tefu double mutant animals also exhibit high levels of apoptosis and fail to induce further apoptosis following irradiation (Oikemus, 2006).
To determine whether the elevated spontaneous apoptosis in these discs requires p53 or mnk (the Drosophila Chk2 homolog), apoptosis was examined in nbs p53 and nbs mnk double mutant discs. The Drosophila p53 and mnk genes are required for induction of apoptosis by X-irradiation. The Drosophila p53 gene was shown to be required for some, but not all, of the apoptosis observed in tefu mutant discs. Apoptosis is substantially reduced in nbs p53 and nbs mnk double mutant discs compared to nbs single mutants. Although p53 has been implicated in a variety of stress response pathways, Chk2 homologs appear to specifically function in DNA damage responses. Thus, these results suggest that the absence of nbs leads to apoptosis via activation of a DNA damage response. This response may be directly activated by unprotected telomeres or by chromosome breaks formed following telomere fusions. The regulation of this response must, however, differ from the regulation of apoptosis 4 h following X-irradiation, which requires wild-type nbs and tefu function (Oikemus, 2006).
DNA damage checkpoint and repair defects in nbs mutant cells: To further compare the function of nbs with tefu, mei-41, and mus304, cell cycle arrest and double-strand DNA break repair were examined. Previous studies have demonstrated that mei-41 is required for G2 arrest at both high (4,000 rads) and low (500 rads) doses of ionizing radiation, whereas tefu is primarily required at low doses, but not high doses. Dose-response curves confirm that mei-41 mutant discs fail to arrest in response to a range of irradiation doses whereas tefu mutant discs have a partial arrest phenotype at low doses, but not at 4,000 rads. Similar to mei-41, nbs is required for cell cycle arrest at all doses tested. These results demonstrate that nbs plays a tefu-independent role in cell cycle arrest and suggests that it acts in the atr-atrip pathway to mediate G2 arrest. A cell cycle arrest defect at low, but not high X-ray doses has been reported for tefu and mre11 mutant cells; however, it was observed that loss of nbs results in an arrest defect at high doses whereas loss of mre11 results in a partial arrest at high doses. nbs mus304 and nbs tefu double mutants also exhibit a cell cycle checkpoint phenotype at high doses; however, the reduced number of mitotic cells and smaller discs indicates that mitosis has been severely disrupted in the double mutants, making direct comparisons to single mutants problematic. It is concluded that nbs, mei-41, and mus304 are all essential for cell cycle arrest at high doses of X-irradiation whereas tefu is not (Oikemus, 2006).
mei-41, mus304, rad50, and mre11 are all required for DNA double-strand break (dsb) repair in Drosophila. The effect of nbs and tefu mutations on dsb formation and repair was examined in metaphase chromosomes from larval neuroblasts. Dsbs can arise as an indirect result of telomere fusion followed by chromosome breakage during mitosis. Broken chromosomes generated by this mechanism will generally retain their centromere. To analyze breaks due to mechanisms other than telomere fusion, the number of acentric chromosome fragments was analyzed. In untreated cells, these fragments may reflect a role in preventing formation of breaks during DNA replication. Both nbs and tefu are required to prevent the spontaneous accumulation of dsbs during normal cell cycles. However, nbs, mre11, mei-41, and mus304 mutant cells all have a more severe phenotype than tefu. Analysis of double mutant cells suggests that nbs and tefu act in parallel to mus304 to prevent accumulation of dsbs. nbs mutant cells also exhibit defective repair of X-irradiation-induced chromosome breaks, consistent with the role of Nbs in the MRN DNA repair complex and with analysis of Drosophila Mre11 and Rad50. Less severe dsb repair defects are seen following X-irradiation of tefu, mei-41, or mus304 mutant cells. Following irradiation, nbs tefu or nbs mus304 double mutant cells do not exhibit a greater defect than nbs single mutants, suggesting that nbs acts in both the tefu and mus304 pathways to mediate repair of induced DNA breaks (Oikemus, 2006).
In summary, nbs acts in both the tefu and mei-41-mus304 DNA damage response pathways. Double mutant analysis indicates that Drosophila nbs acts in common genetic pathways with tefu and mus304 during DNA repair. In addition, nbs has DNA damage response phenotypes in common with both tefu (defective induction of apoptosis) and mei-41-mus304 (defective induction of cell cycle arrest at high doses of X-irradiation). Although Nbs1 homologs are best known for their roles in DNA repair and signaling in the ATM pathway, human Nbs1 is also required for signaling by the ATR pathway. Thus, nbs has a conserved role in ATM- and ATR-ATRIP-dependent DNA damage responses (Oikemus, 2006).
Two DNA damage response pathways contribute to telomere protection: Metaphase larval neuroblasts were also used to examine the roles of different DNA damage response genes in telomere protection. Previous studies have demonstrated that Drosophila tefu, mre11, and rad50 mutant cells have a partial defect in telomere protection. Consistent with these results, nbs mutant animals exhibit a high frequency of cells with one or more fusions. These fusions are observed during both metaphase and anaphase. nbs tefu double mutant cells exhibit similar fusion rates as tefu single mutants, indicating that these genes act in a common telomere protection pathway. These results are consistent with results in Drosophila and other organisms, indicating that ATM and components of the MRN complex act in a common telomere protection pathway. Downstream targets of ATM in the mammalian DNA damage response pathway include Nbs1 and the checkpoint kinases CHK1 and CHK2. The Drosophila homologs of these kinases are encoded by the grp and mnk genes and are required for DNA damage-induced apoptosis and cell cycle arrest. Both telomere protection and chromosome break repair are normal in grp mnk double mutant cells, indicating that other targets of Drosophila tefu and nbs are responsible for their telomere protection and DNA repair functions (Oikemus, 2006).
Compared with mutations in the genes that encode the telomere protection proteins HOAP and HP1, mutations in tefu, nbs, mre11, and rad50 exhibit a significantly lower frequency of telomere fusions, indicating that there may be a tefu-nbs-independent pathway for telomere protection. In mammals, the ATR checkpoint kinase is recruited to sites of DNA damage by ATRIP and acts in parallel to the ATM kinase in the DNA damage response. In budding and fission yeast, disruption of both atm/atr homologs results in loss of telomere protection. Mutations in mei-41 or mus304 do not result in telomere protection defects. However, nbs mus304, tefu mus304, and tefu mei-41 double mutant animals all show higher rates of telomere fusion than the corresponding single mutants indicating that the mei-41-mus304 pathway acts in parallel to a tefu-nbs pathway to mediate telomere protection. The higher fusion frequency in tefu mei-41 double mutants compared with tefu mus304 double mutants may indicate that there is a small amount of mei-41 activity in the absence of mus304 (Oikemus, 2006).
DNA damage response genes regulate telomeric HOAP: The formation of telomere fusions requires two steps: (1) the failure of telomeric protein complexes, such as HP1-HOAP, to prevent telomeric DNA ends from being recognized as damage-induced ends and (2) the subsequent ligation of unprotected telomeres by DNA repair systems. To probe the role of DNA damage response pathways in the first step, HOAP localization was examined in individual mitotic cells (neuroblasts). Previously, it was shown that levels of the telomere protection proteins HP1 and HOAP are reduced at the telomeres of polytene chromosomes from tefu salivary gland cells, but that telomeric HOAP is not strongly reduced in mitotic chromosomes from neuroblasts; these results suggest that in the absence of tefu, neuroblasts utilize an alternative mechanism for HOAP localization. In contrast, both salivary glands and neuroblasts required mre11 and rad50 for normal HOAP localization (Oikemus, 2006).
The frequency of neuroblast telomeres with HOAP staining and the intensity of staining at those telomeres were examined in wild-type and mutant cells. Measurements of fluorescence intensity can be used to demonstrate that HOAP levels at individual telomeres are reproducibly increased or decreased in different genotypes. (However, it is noted that there may not be a linear relationship between the percent change of fluorescence observed and the percent change of telomeric HOAP protein levels.) Most wild-type, tefu, or mus304 mutant metaphase cells are HOAP positive; between 77% and 94% of these cells had HOAP signals at chromosome ends. Among the HOAP positive cells, between 66% and 72% of telomeres stained for HOAP. The fluorescence intensity of HOAP staining was similar at the telomeres of each of the major chromosome arms in both wild-type and mus304 mutant cells. However, the average intensity of HOAP staining was elevated in tefu mutant cells, indicating that although HOAP is still recruited to telomeres, the mechanism regulating HOAP levels at telomeres may be perturbed. A more severe effect on HOAP localization was observed in nbs mutant metaphases, with only 44% of metaphases displaying HOAP signals and only 30% of the telomeres in those cells staining for HOAP. This phenotype is similar to that reported for mre11 and rad50 mutant neuroblasts. At the few HOAP-positive telomeres that are present in nbs mutant cells, HOAP fluorescence staining intensity was elevated compared to wild type, similar to the HOAP staining at tefu mutant telomeres. Together with the genetic data indicating that tefu and nbs act in a common telomere protection pathway, these results suggest that an alternative pathway can maintain HOAP levels at telomeres, but that this pathway is much less efficient in nbs mutant cells (Oikemus, 2006).
Quantification of HOAP telomeric signals: Since mus304 nbs, mus304 tefu, and mei-41 tefu double mutant cells have more severe telomere fusion phenotypes than nbs or tefu single mutants, the mei-41-mus304 pathway is a clear candidate to recruit HOAP to telomeres in the absence of tefu or nbs. mus304 single mutant animals do not exhibit a defect in either the frequency of HOAP-positive telomeres or the intensity of HOAP staining at those telomeres. In contrast, no telomeric HOAP staining was detected in mus304 tefu or mus304 nbs double mutant cells. Thus, the mei-41-mus304 pathway partially compensates for the absence of tefu, limiting the severity of the tefu telomere fusion phenotype. Cells lacking both pathways exhibit loss of telomeric HOAP and a severe telomere fusion phenotype. In a report published while this work was in preparation, Bi (2005) also found that disruption of the Drosophila atm and atr pathways results in a high frequency of telomere fusions and loss of telomeric HOAP (Oikemus, 2006).
These results support the model that the Drosophila tefu and mei-41-mus304 DNA damage response pathways mediate telomere protection by recruiting or maintaining HOAP at telomeres. The more severe HOAP localization phenotype of nbs mutant cells compared with tefu mutant cells indicates that nbs has a tefu-independent role in telomere protection. As described above, the common DNA repair and damage response phenotypes of nbs with mei-41 and mus304 indicate that nbs also acts in the mei-41-mus304 DNA damage response pathway. Thus, one explanation for the lower frequency of HOAP positive telomeres in nbs compared to tefu mutant cells is that nbs mutations both disrupt the tefu telomere protection function and partially disable a compensatory telomere protection pathway mediated by mei-41-mus304 (Oikemus, 2006).
There is a good correlation between the levels of telomeric HOAP and the frequency of telomere fusion, except in nbs, mre11, and rad50 mutants. In yeast and mammalian cells, some DNA repair genes are required to both maintain telomere protection and to promote joining of unprotected telomeres. The observed telomere fusion frequency in nbs mutant cells may reflect the combined effects of decreased telomere protection and inefficient fusion of unprotected telomeres. Although the loss of nbs has a more severe effect than tefu on telomeric HOAP, nbs and tefu mutant cells have similar telomere fusion frequencies. nbs mutations have a more severe effect on repair of DNA breaks, suggesting that nbs mutant cells may also have reduced joining of unprotected telomeres. Consistent with a role for nbs in fusion of unprotected telomeres, nbs mus304 mutant cells have a lower telomere fusion frequency than tefu mus304 mutant cells, despite undetectable levels of telomeric HOAP in both genotypes. Similarly, nbs cav double mutant cells have a lower telomere fusion frequency than tefu cav double mutant cells (Oikemus, 2006).
In summary, DNA damage response genes are essential for the telomeric localization of the protection protein HOAP. Analysis of DNA repair, telomere fusions and HOAP localization suggests that the telomere fusion frequency reflects a combination of defective protection and reduced fusion of unprotected chromosomes. Although these results do not rule out the possibility that DNA damage response genes are also required for modification of HP1 and HOAP complexes at telomeres, they strongly suggest that recruitment or maintenance of these complexes to telomeres is critical for telomere protection (Oikemus, 2006).
DNA damage response pathways are required for epigenetic protection of telomeres: In many organisms, telomere-specific sequences are required to recruit proteins that prevent chromosome end fusion. Loss of these sequences in cells that do not express telomerase or that are mutant for DNA damage response genes can result in telomere fusions. However, in Drosophila, the stable protection of terminally deleted chromosomes from telomere fusion suggests that a sequence-independent mechanism acts to protect the telomeres of normal chromosomes. Given the requirement of the tefu and mei-41-mus304 DNA damage response pathways for telomere protection, it is proposef that recognition of chromosome ends contributes to this epigenetic phenomenon. One prediction of this model is that cells lacking these pathways will exhibit telomere fusion without loss of telomeric DNA sequences such as HeT-A. HeT-A sequences should not be lost simply as a secondary effect of unprotected telomeres since telomere fusions in cells lacking HP1 function still retain these sequences. A second prediction is that terminal deletion chromosomes lacking telomeric sequences will still fuse in the absence of the DNA damage response pathways. This observation would rule out the possibility that the epigenetic mechanism for protection of terminal deletions utilizes an alternative mechanism to recruit HP1 and HOAP that is independent of the DNA damage response pathways. These predictions can be evaluated in animals with the extreme telomere fusion phenotype associated with loss of both the tefu and mei-41-mus304 DNA damage response pathways (Oikemus, 2006).
To test the first prediction, the telomere-specific retrotransposon HeT-A was analyzed at individual telomeres of DNA damage response defective cells by fluorescence in situ hybridization. Measurements of fluorescence intensity can be used to demonstrate that HeT-A levels at individual telomeres are reproducibly increased or decreased in different genotypes. (However, it is noted that there may not be a linear relationship between the percent change of fluorescence observed and the percent change of telomeric HeT-A DNA.) Previously, telomere fusions in tefu mutant cells were shown to retain at least some HeT-A sequences. However, these studies only examined mutants with mild telomere fusion phenotypes and were less thorough than the analysis presented in this study. Because the number of HeT-A copies per telomere can vary between strains, particularly in strains with altered HP1 function, HeT-A signals at free chromosome ends in homozygous mutant animals were compared to chromosome fusion sites in the same cells and to free chromosome ends in an appropriate heterozygous parental strain. HeT-A is still present at free telomeres and at chromosome fusion sites in tefu, nbs, tefu mus304, and nbs mus304 homozygous mutant cells. For each genotype, both the frequency and intensity of HeT-A staining at chromosome fusions is equal to or greater than that observed at the free chromosome ends, indicating that loss of telomere-specific sequences does not correlate with telomere fusion in cells with defective DNA damage response pathways. Note that if a HeT-A-positive telomere fuses with another HeT-A-positive telomere, the intensity of staining will increase; if it fuses with a HeT-A-negative telomere or a chromosome break, the intensity should be the same. Different genotypes exhibit different relative intensities of HeT-A staining at chromosome fusions compared to free ends. These differences may reflect different frequencies of telomere-telomere fusions versus telomere-break fusions or differences in the precise mechanism of telomere fusion. Nonetheless, the observation that the staining intensity at fusions is equal to or greater than the intensity at free chromosome ends demonstrates that loss of these sequences is not required for fusion in any of these genotypes (Oikemus, 2006).
Percent chromosomes with HeT-A staining: The frequency and intensity of HeT-A staining was also compared at the free chromosome ends of mutant cells and the corresponding parental strain. The frequency of HeT-A staining at chromosome ends in homozygous nbs, nbs mus304, and tefu mus304 mutant cells is lower than in cells from the corresponding heterozygous strains. Although this decrease could reflect removal of telomeric sequences in homozygous mutant animals, two other factors are likely to contribute. First, defective DNA repair generates chromosome ends without telomeric sequences. Several of these mutations result in high levels of spontaneous breaks. Second, progression of cells with telomere fusions through mitosis generates anaphase bridges and chromosome breaks via the fusion/bridge/break cycle. In one example, an internal site of HeT-A (the original fusion site) was adjacent to a chromosome end without HeT-A (the break site). Chromosome ends with adjacent internal HeT-A sites are found in all mutant cells with telomere fusions. The overall frequency of breaks resulting from fusion is underestimated by this analysis since some broken chromosomes will not include the original fusion site. Thus, chromosome breaks can account for the increased number of ends without HeT-A staining. However, at those chromosome ends that are HeT-A positive, the intensity of staining is equal to or greater than in the corresponding heterozygous cells. Combined with the analysis of HeT-A staining at fusion sites, these results indicate that the fusion phenotype of single or double mutants in the DNA damage response pathways is not due to loss of telomeric sequences (Oikemus, 2006).
A second prediction of the end-recognition model for Drosophila telomere protection is that both normal and terminally deleted chromosomes will exhibit similar frequencies of fusion in cells lacking the DNA damage response pathways. The stable protection of terminally deleted chromosomes in wild-type cells suggests that the telomeres of normal chromosomes are also protected by sequence-independent mechanism; however, it is also possible that terminally deleted chromosomes acquire an alternative mechanism for telomere protection and that the DNA damage response pathways must act in conjunction with a sequence-specific mechanism. To address this possibility, fusion rates of a normal and a terminally deleted X chromosome were examined in tefu mus304 double mutant cells. Previous experiments have demonstrated that the telomere protection gene UbcD1 is required to prevent fusion of terminally deleted chromosomes. In tefu mus304 double mutant cells, a normal and a terminally deleted X chromosome fused to the sister or to heterologous chromosomes at a high frequency. The fusion frequency is similar but lower than observed with a normal X chromosome; this difference may indicate that the terminally deleted chromosome is slightly less sensitive to the loss of DNA damage signaling pathways. Nonetheless, the frequent fusion of terminally deleted chromosomes in tefu mus304 double mutant cells directly demonstrates that the DNA damage response pathways act in an epigenetic mechanism for telomere protection (Oikemus, 2006).
It is concluded DNA damage response genes have evolutionarily conserved roles in telomere function. Unprotected telomeres are recognized by these pathways and elicit a variety of cellular responses including apoptosis and end-to-end fusion of chromosomes. However, these same pathways are also required to promote telomere protection. The Drosophila atm and atr-atrip DNA damage response pathways act in an epigenetic mechanism to mediate telomere protection. In cells lacking both pathways, the chromatin-associated protein HOAP is not recruited to telomeres and both normal and terminally deleted chromosomes undergo fusion at a high frequency. Furthermore, fusion of normal telomeres occurs without loss of telomere-specific sequences. Taken together, these results support an end-recognition model in which DNA damage response proteins recognize a DNA structure at the chromosome end and recruit or stabilize the telomere protection proteins HP1 and HOAP at telomeres; in turn, these proteins act to prevent the ligation of chromosome ends by DNA repair enzymes and the activation of p53-dependent apoptosis. In other organisms, a similar epigenetic mechanism may act in conjunction with sequence-specific protection mechanisms or may be utilized to promote protection of critically short telomeres, which are least able to utilize sequence-specific binding proteins (Oikemus, 2006).
Nijmegen breakage syndrome (NBS) is an autosomal recessive disorder characterized by increased cancer incidence, cell cycle checkpoint defects, and ionizing radiation sensitivity. The gene encoding p95, a member of the hMre11/hRad50 double-strand break repair complex, has been isolated. The p95 gene mapped to 8q21.3, the region that contains the NBS locus, and p95 was absent from NBS cells established from NBS patients. p95 deficiency in these cells completely abrogates the formation of hMre11/hRad50 ionizing radiation-induced foci. Comparison of the p95 cDNA to the NBS1 cDNA indicated that the p95 gene and NBS1 are identical. The implication of hMre11/hRad50/p95 protein complex in NBS reveals a direct molecular link between DSB repair and cell cycle checkpoint functions (Carney, 1998).
NBS is an autosomal recessive chromosomal instability syndrome characterized by microcephaly, growth retardation, immunodeficiency, and cancer predisposition. Cells from NBS patients are hypersensitive to ionizing radiation with cytogenetic features indistinguishable from ataxia telangiectasia. This study describes the positional cloning of a gene encoding a novel protein, nibrin. It contains two modules found in cell cycle checkpoint proteins, a forkhead-associated domain adjacent to a breast cancer carboxy-terminal domain. A truncating 5 bp deletion was identified in the majority of NBS patients, carrying a conserved marker haplotype. Five further truncating mutations were identified in patients with other distinct haplotypes. The domains found in nibrin and the NBS phenotype suggest that this disorder is caused by defective responses to DNA double-strand breaks (Varon, 1998)
NBS, a chromosomal instability disorder, is characterized in part by cellular hypersensitivity to ionizing radiation. Repair of DNA double-strand breaks by radiation is dependent on a multifunctional complex containing Rad50, Mre11, and the NBS1 gene product, p95 (NBS protein, nibrin). The role of p95 in these repair processes is unknown. This study demonstrates that Mre11 is hyperphosphorylated in a cell cycle-independent manner in response to treatment of cells with genotoxic agents including gamma irradiation. This response is abrogated in two independently established NBS cell lines that have undetectable levels of the p95 protein. NBS cells are also deficient for radiation-induced nuclear foci containing Mre11, while those with Rad51 are unaffected. An analysis of the kinetic relationship between Mre11 phosphorylation and the appearance of its radiation-induced foci indicates that the former precedes the latter. Together, these data suggest that specific phosphorylation of Mre11 is induced by DNA damage, and p95 is essential in this process, perhaps by recruiting specific kinases (Dong, 1999).
The Mre11.Rad50.nibrin protein complex plays an essential role in the mammalian cellular response to DNA double-strand breaks. The disorder Nijmegen breakage syndrome (NBS) results from mutations in the NBS1 gene that encodes nibrin, and NBS cells are radiosensitive and defective in S-phase checkpoint activation following irradiation. In response to radiation, nibrin is phosphorylated by Atm, and the Mre11.Rad50.nibrin complex relocalizes to form punctate nuclear foci. The N terminus of nibrin contains a forkhead-associated (FHA) domain and a breast cancer C-terminal (BRCT) domain, the functions of which are unclear. To determine the role of the FHA and BRCT domains in nibrin function, site-directed mutagenesis of conserved residues in these motifs was performed. Mutations in the nibrin FHA and BRCT domains did not affect interaction with Mre11.Rad50 or nuclear localization of the complex. However, mutation of conserved residues in either domain disrupted nuclear focus formation and blocked nibrin phosphorylation after irradiation, suggesting that these events may be functionally interdependent. Despite an effect on nibrin phosphorylation, expression of the FHA or BRCT mutants in NBS cells restored the downstream phosphorylation of Chk2 and Smc1, necessary for S-phase checkpoint activation. None of the mutations revealed separate functions for the FHA or BRCT domains, suggesting they do not function independently (Cerosaletti, 2003).
The human Rad50/Mre11/Nbs1 complex (hR/M/N) functions as an essential guardian of genome integrity by directing the proper processing of DNA ends, including DNA breaks. This biological function results from its ability to tether broken DNA molecules. hR/M/N's dynamic molecular architecture consists of a globular DNA-binding domain from which two 50-nm-long coiled coils protrude. The coiled coils are flexible and their apices can self-associate. The flexibility of the coiled coils allows their apices to adopt an orientation favourable for interaction. However, this also allows interaction between the tips of two coiled coils within the same complex, which competes with and frustrates the intercomplex interaction required for DNA tethering. This study shows that the dynamic architecture of hR/M/N is markedly affected by DNA binding. DNA binding by the hR/M/N globular domain leads to parallel orientation of the coiled coils; this prevents intracomplex interactions and favours intercomplex associations needed for DNA tethering. The hR/M/N complex thus is an example of a biological nanomachine in which binding to its ligand, in this case DNA, affects the functional conformation of a domain located 50 nm distant (Moreno-Herrero, 2005).
The hR/M complex is a heterotetramer, R2M2, arranged with a globular DNA-binding domain, including the Mre11 dimer and the two Rad50 ATPase domains, from which the long intramolecular coiled coils of Rad50 protrude. The coiled-coil apex contains a CXXC amino-acid motif that forms a structure described as a zinc hook. Genetic experiments in Saccharomyces cerevisiae have shown that the zinc hook is an important determinant of Rad50 function. Two CXXC motifs can dimerize by the coordination of a Zn2þ ion, providing a possible interface for interaction between hR/M complexes. hR/M complexes form oligomers on linear DNA where interactions between the apices of the coiled coils then tether DNA molecules. Conformational changes that alter the orientation, flexibility or dynamics of the coiled coils could be exploited to control intercomplex versus intracomplex interaction of the coiled-coil apices and therefore the biological function of hR/M (Moreno-Herrero, 2005 and references therein).
The hR/M complex can also include Nbs1. The Nbs1 component probably interacts with the globular domain of the R/M complex and functions in signalling the presence of DNA breaks to the cell cycle checkpoint machinery. The three-component complex hR/M/N appeared very similar to hR/M in the atomic force microscopy study. The presence of the third subunit was evident as a larger globular domain with an average height of ~8.0 nm. In the absence of DNA, the hR/M/N complex also has bent, open coiled coils with apices in open and closed conformations. After DNA binding, in the presence of a molar excess of a 90-bp oligonucleotide, the coiled coils of hR/M/N also adopt a parallel conformation. The use of short DNA oligonucleotides for binding prevented the occurrence of large hR/M/N oligomers and thus allowed single intercomplex interactions to be observed through the apices of the coiled coils. Indeed, the intercomplex interaction of coiled-coil apices necessary for tethering is occasionally observed in these conditions. Such an intercomplex configuration was never observed for hR/M and hR/M/N in the absence of DNA (Moreno-Herrero, 2005).
The existing molecular picture of hR/M/N function involves three processes: first, binding of the complex to DNA ends; second, the formation of large DNA-bound oligomers with the Rad50 apices of the coiled coils protruding; and third, subsequent interaction between the coiled coils of hR/M/N complexes bound to different DNA ends, to tether them and keep them close in nuclear space. Interaction between the apices of the coiled coils is therefore essential for R/M/N function. A conundrum of this molecular description centers on the control of this interaction. A single hR/M/N complex has two such apices in high local concentration. Because of the flexibility of the Rad50 coiled coils these can, and do, interact. DNA-bound hR/M/N oligomers also have many apices in high local concentration, and interaction between those bound to the same DNA molecule would be futile for tethering independent DNA ends. This study has shown that this futile intracomplex joining of Rad50 coiled-coil apices is prevented once DNA is engaged. The hR/M/N coiled coils become oriented in such a way that intracomplex interactions are prevented, while at the same time intercomplex interactions needed for tethering are favoured (Moreno-Herrero, 2005).
ATM has a central role in controlling the cellular responses to DNA damage. It and other phosphoinositide 3-kinase-related kinases (PIKKs) have giant helical HEAT repeat domains in their amino-terminal regions. The functions of these domains in PIKKs are not well understood. ATM activation in response to DNA damage appears to be regulated by the Mre11-Rad50-Nbs1 (MRN) complex, although the exact functional relationship between the MRN complex and ATM is uncertain. Two pairs of HEAT repeats in fission yeast ATM (Tel1) interact with an FXF/Y motif at the C terminus of Nbs1. This interaction resembles nucleoporin FXFG motif binding to HEAT repeats in importin-beta. Budding yeast Nbs1 (Xrs2) appears to have two FXF/Y motifs that interact with Tel1 (ATM). In Xenopus egg extracts, the C terminus of Nbs1 recruits ATM to damaged DNA, where it is subsequently autophosphorylated. This interaction is essential for ATM activation. A C-terminal 147-amino-acid fragment of Nbs1 that has the Mre11- and ATM-binding domains can restore ATM activation in an Nbs1-depleted extract. It is concluded that an interaction between specific HEAT repeats in ATM and the C-terminal FXF/Y domain of Nbs1 is essential for ATM activation. It is proposed that conformational changes in the MRN complex that occur upon binding to damaged DNA are transmitted through the FXF/Y-HEAT interface to activate ATM. This interaction also retains active ATM at sites of DNA damage (You, 2005).
Human Nbs1 and its homolog Xrs2 in Saccharomyces cerevisiae are part of the conserved MRN complex (MRX in yeast) which plays a crucial role in maintaining genomic stability. NBS1 corresponds to the gene mutated in the Nijmegen breakage syndrome (NBS) known as a radiation hyper-sensitive disease. Despite the conservation and the importance of the MRN complex, the high sequence divergence between Nbs1 and Xrs2 precluded the identification of common domains downstream of the N-terminal Fork-Head Associated (FHA) domain. Using HMM-HMM profile comparisons and structure modelling, the existence of a tandem BRCT was assessed in both Nbs1 and Xrs2 after the FHA. The structure-based conservation analysis of the tandem BRCT in Nbs1 supports its function as a phosphoserine binding domain. Remarkably, the 5 bp deletion observed in 95% of NBS patients cleaves the tandem at the linker region while preserving the structural integrity of each BRCT domain in the resulting truncated gene products (Becker, 2006).
The c-myc proto-oncogene encodes a ubiquitous transcription factor involved in the control of cell growth and implicated in inducing tumorigenesis. Understanding the function of c-Myc and its role in cancer depends upon the identification of c-Myc target genes. Nijmegen breakage syndrome (NBS) is a chromosomal-instability syndrome associated with cancer predisposition, radiosensitivity, and chromosomal instability. The NBS gene product, NBS1 (p95 or nibrin), is a part of the hMre11 complex, a central player associated with double-strand break (DSB) repair. NBS1 contains domains characteristic for proteins involved in DNA repair, recombination, and replication. This study shows that c-Myc directly activates NBS1. c-Myc-mediated induction of NBS1 gene transcription occurs in different tissues, is independent of cell proliferation, and is mediated by a c-Myc binding site in the intron 1 region of NBS1 gene. Overexpression of NBS1 in Rat1a cells increased cell proliferation. These results indicate that NBS1 is a direct transcriptional target of c-Myc and links the function of c-Myc to the regulation of DNA DSB repair pathway operating during DNA replication (Chiang, 2003).
Telomeres allow cells to distinguish natural chromosome ends from damaged DNA and protect the ends from degradation and fusion. In human cells, telomere protection depends on the TTAGGG repeat binding factor, TRF2, which has been proposed to remodel telomeres into large duplex loops (t-loops). This study shows by nanoelectrospray tandem mass spectrometry that RAD50 protein is present in TRF2 immunocomplexes. Protein blotting showed that a small fraction of RAD50, MRE11 and the third component of the MRE11 double-strand break (DSB) repair complex, the Nijmegen breakage syndrome protein (NBS1), is associated with TRF2. Indirect immunofluorescence demonstrated the presence of RAD50 and MRE11 at interphase telomeres. NBS1 was associated with TRF2 and telomeres in S phase, but not in G1 or G2. Although the MRE11 complex accumulates in irradiation-induced foci (IRIFs) in response to gamma-irradiation, TRF2 does not relocate to IRIFs and irradiation does not affect the association of TRF2 with the MRE11 complex, arguing against a role for TRF2 in DSB repair. Instead, it is proposed that the MRE11 complex functions at telomeres, possibly by modulating t-loop formation (Zhu, 2000).
Hypomorphic mutations which lead to decreased function of the NBS1 gene are responsible for Nijmegen breakage syndrome, a rare autosomal recessive hereditary disorder that imparts an increased predisposition to development of malignancy. The NBS1 protein is a component of the MRE11/RAD50/NBS1 complex that plays a critical role in cellular responses to DNA damage and the maintenance of chromosomal integrity. Using small interfering RNA transfection, NBS1 protein levels were knocked down and relevant phenotypes were analyzed in two closely related human lymphoblastoid cell lines with different p53 status, namely wild-type TK6 and mutated WTK1. Both TK6 and WTK1 cells showed an increased level of ionizing radiation-induced mutation at the TK and HPRT loci, impaired phosphorylation of H2AX (gamma-H2AX), and impaired activation of the cell cycle checkpoint regulating kinase, Chk2. In TK6 cells, ionizing radiation-induced accumulation of p53/p21 and apoptosis were reduced. There was a differential response to ionizing radiation-induced cell killing between TK6 and WTK1 cells after NBS1 knockdown; TK6 cells were more resistant to killing, whereas WTK1 cells were more sensitive. NBS1 deficiency also resulted in a significant increase in telomere association that was independent of radiation exposure and p53 status. These results provide the first experimental evidence that NBS1 deficiency in human cells leads to hypermutability and telomere associations, phenotypes that may contribute to the cancer predisposition seen among patients with this disease (Zhang, 2005).
A central function of telomeres is to prevent chromosome ends from being recognized as DNA double-strand breaks (DSBs). Several proteins involved in processing DSBs associate with telomeres, but the roles of these factors at telomeres are largely unknown. To investigate whether the Mre11/Rad50/Nbs1 (MRN) complex is involved in the generation of proper 3' G-overhangs at human telomere ends, RNA interference was used to decrease expression of MRN and the effects were analyzed. Reduction of MRN resulted in a transient shortening of G-overhang length in telomerase-positive cells. The terminal nucleotides of both C- and G-rich strands remain unaltered in Mre11-diminished cells, indicating that MRN is not responsible for specifying the final end-processing event. The reduction in overhang length was not seen in telomerase-negative cells, but was observed after the expression of exogenous telomerase, which suggested that the MRN complex might be involved in the recruitment or action of telomerase (Chai, 2006).
The Atm protein kinase and Mre11-Rad50-nibrin (MRN) complex play an integral role in the cellular response to DNA double-strand breaks. Mutations in Mre11 and nibrin result in the radiosensitivity disorders ataxia-telangiectasia-like disorder (ATLD) and Nijmegen breakage syndrome (NBS), respectively. Cells from ATLD and NBS patients are deficient in activation of the Atm protein kinase and phosphorylation of downstream Atm targets following irradiation. However, the roles of individual MRN complex proteins in Atm function are not clear, because the mutations in NBS and ATLD cells result in global effects on the MRN complex. The C-terminal 100 amino acids of nibrin are necessary and sufficient to translocate the MRN complex to the nucleus. Advantage was taken of this feature of nibrin to create isogenic cell lines lacking either nibrin or Mre11-Rad50 in the nucleus. It was found that nuclear expression of Mre11-Rad50, but not nibrin, stimulates Atm activation at early times after low doses of radiation. At later times or higher doses of irradiation, Atm activation is independent of Mre11-Rad50 or nibrin. The requirement of MRN complex proteins for downstream Atm phosphorylation events following irradiation is more complex. Phosphorylation of nibrin and Chk2 by Atm requires Mre11-Rad50 expression in the nucleus at early times after irradiation, reflecting the stimulation of Atm activation by Mre11-Rad50. By contrast, autophosphorylation of Chk2 and phosphorylation of Smc1 at Ser-957 is dependent on the MRN complex 60 min after irradiation, even though Atm is activated at that time point. These results indicate an independent role for Mre11-Rad50 in the activation of Atm and suggest nibrin and/or Mre11-Rad50 also act as adaptors for some downstream Atm phosphorylation events (Cerosaletti, 2004).
The function has been examined of the human ortholog of Saccharomyces cerevisiae Rif1 (Rap1-interacting factor 1). Yeast Rif1 associates with telomeres and regulates their length. In contrast, human Rif1 does not accumulate at functional telomeres, but localizes to dysfunctional telomeres and to telomeric DNA clusters in ALT cells, a pattern of telomere association typical of DNA-damage-response factors. After induction of double-strand breaks (DSBs), Rif1 forms foci that colocalize with other DNA-damage-response factors. This response is strictly dependent on ATM and 53BP1, but not affected by diminished function of ATR (ATM- and Rad3-related kinase), BRCA1, Chk2, Nbs1, and Mre11. Rif1 inhibition results in radiosensitivity and a defect in the intra-S-phase checkpoint. The S-phase checkpoint phenotype is independent of Nbs1 status, arguing that Rif1 and Nbs1 act in different pathways to inhibit DNA replication after DNA damage. These data reveal that human Rif1 contributes to the ATM-mediated protection against DNA damage and point to a remarkable difference in the primary function of this protein in yeast and mammals (Silverman, 2004).
The DNA damage response, triggered by DNA replication stress or DNA damage, involves the activation of DNA repair and cell cycle regulatory proteins including the MRN (Mre11, Rad50, and Nbs1) complex and replication protein A (RPA). The induction of replication stress by hydroxyurea (HU) or DNA damage by camptothecin (CAMPT), etoposide (ETOP), or mitomycin C (MMC) led to the formation of nuclear foci containing phosphorylated Nbs1. HU and CAMPT treatment also led to the formation of RPA foci that co-localized with phospho-Nbs1 foci. After ETOP treatment, phospho-Nbs1 and RPA foci are detected but not within the same cell. MMC treatment resulted in phospho-Nbs1 foci formation in the absence of RPA foci. Consistent with the presence or absence of RPA foci, RPA hyperphosphorylation was present following HU, CAMPT, and ETOP treatment but absent following MMC treatment. The lack of co-localization of phospho-Nbs1 and RPA foci may be due to relatively shorter stretches of single-stranded DNA generated following ETOP and MMC treatment. These data suggest that, even though the MRN complex and RPA can interact, their interaction may be limited to responses to specific types of lesions, particularly those that have longer stretches of single-stranded DNA. In addition, the consistent formation of phospho-Nbs1 foci in all of the treatment groups suggests that the MRN complex may play a more universal role in the recognition and response to DNA lesions of all types, whereas the role of RPA may be limited to certain subsets of lesions (Robison, 2005).
ATM and ATR are two related kinases essential for signalling DNA damage. Although ATM is thought to be the principle kinase responsible for signalling ionising radiation (IR)-induced DNA damage, ATR also contributes to signalling this form of genotoxic stress. However, the molecular basis of differential ATM and ATR activation in response to IR remains unclear. This study reports that ATR is recruited to sites of IR-induced DNA damage significantly later than activation of ATM. ATR is recruited to IR-induced nuclear foci in G(1) and S phase of the cell cycle, supporting a role for ATR in detecting DNA damage outside of S phase. In addition, recruitment of ATR to sites of IR-induced DNA damage is concomitant with appearance of large tracts of single-stranded DNA (ssDNA) and that this event is dependent on ATM and components of the Mre11/Rad50/Nbs1 (MRN) protein complex (Adams, 2006).
Ataxia-telangiectasia (A-T) and Nijmegen breakage syndrome (NBS) are recessive genetic disorders with susceptibility to cancer and similar cellular phenotypes. The protein product of the gene responsible for A-T, designated ATM, is a member of a family of kinases characterized by a carboxy-terminal phosphatidylinositol 3-kinase-like domain. The NBS1 protein is specifically mutated in patients with Nijmegen breakage syndrome and forms a complex with the DNA repair proteins Rad50 and Mrel1. Phosphorylation of NBS1, induced by ionizing radiation, requires catalytically active ATM. Complexes containing ATM and NBS1 exist in vivo in both untreated cells and cells treated with ionizing radiation. Two residues of NBS1, Ser 278 and Ser 343, were identified that are phosphorylated in vitro by ATM and whose modification in vivo is essential for the cellular response to DNA damage. This response includes S-phase checkpoint activation, formation of the NBS1/Mrel1/Rad50 nuclear foci and rescue of hypersensitivity to ionizing radiation. Together, these results demonstrate a biochemical link between cell-cycle checkpoints activated by DNA damage and DNA repair in two genetic diseases with overlapping phenotypes (Zhao, 2000).
The ATM protein kinase is a primary activator of the cellular response to DNA double-strand breaks (DSBs). In response to DSBs, ATM is activated and phosphorylates key players in various branches of the DNA damage response network. ATM deficiency causes the genetic disorder ataxia-telangiectasia (A-T), characterized by cerebellar degeneration, immunodeficiency, radiation sensitivity, chromosomal instability and cancer predisposition. The MRN complex, whose core contains the Mre11, Rad50 and Nbs1 proteins, is involved in the initial processing of DSBs. Hypomorphic mutations in the NBS1 and MRE11 genes lead to two other genomic instability disorders: the Nijmegen breakage syndrome (NBS) and A-T like disease (A-TLD), respectively. The order in which ATM and MRN act in the early phase of the DSB response is unclear. This study shows that functional MRN is required for ATM activation, and consequently for timely activation of ATM-mediated pathways. Collectively, these and previous results assign to components of the MRN complex roles upstream and downstream of ATM in the DNA damage response pathway and explain the clinical resemblance between A-T and A-TLD (Uziel, 2003).
The complex containing the Mre11, Rad50, and Nbs1 proteins (MRN) is essential for the cellular response to DNA double-strand breaks, integrating DNA repair with the activation of checkpoint signaling through the protein kinase ATM. MRN stimulates the kinase activity of ATM in vitro toward its substrates p53, Chk2, and histone H2AX. MRN makes multiple contacts with ATM and appears to stimulate ATM activity by facilitating the stable binding of substrates. Phosphorylation of Nbs1 is critical for MRN stimulation of ATM activity toward Chk2, but not p53. Kinase-deficient ATM inhibits wild-type ATM phosphorylation of Chk2, consistent with the dominant-negative effect of kinase-deficient ATM in vivo (Lee, 2004).
The ataxia-telangiectasia mutated (ATM) kinase signals the presence of DNA double-strand breaks in mammalian cells by phosphorylating proteins that initiate cell-cycle arrest, apoptosis, and DNA repair. The Mre11-Rad50-Nbs1 (MRN) complex acts as a double-strand break sensor for ATM and recruits ATM to broken DNA molecules. Inactive ATM dimers were activated in vitro with DNA in the presence of MRN, leading to phosphorylation of the downstream cellular targets p53 and Chk2. ATM autophosphorylation is not required for monomerization of ATM by MRN. The unwinding of DNA ends by MRN is essential for ATM stimulation, consistent with the central role of single-stranded DNA as an evolutionarily conserved signal for DNA damage (J.-H. Lee, 2005).
The Atm protein kinase is central to the DNA double-strand break response in mammalian cells. After irradiation, dimeric Atm undergoes autophosphorylation at Ser 1981 and dissociates into active monomers. Atm activation is stimulated by expression of the Mre11/Rad50/nibrin complex. A C-terminal fragment of nibrin, containing binding sites for both Mre11 and Atm, is sufficient to provide this stimulatory effect in Nijmegen breakage syndrome (NBS) cells. To discriminate whether nibrin's role in Atm activation is to bind and translocate Mre11/Rad50 to the nucleus or to interact directly with Atm, an Mre11 transgene with a C-terminal NLS sequence was expressed in NBS fibroblasts. The Mre11-NLS protein complexes with Rad50, localizes to the nucleus in NBS fibroblasts, and associates with chromatin. However, Atm autophosphorylation is not stimulated in cells expressing Mre11-NLS, nor are downstream Atm targets phosphorylated. To determine whether nibrin-Atm interaction is necessary to stimulate Atm activation, nibrin transgenes lacking the Atm binding domain were expressed in NBS fibroblasts. The nibrin DeltaAtm protein interacted with Mre11/Rad50; however, Atm autophosphorylation is dramatically reduced after irradiation in NBS cells expressing the nibrin DeltaAtm transgenes relative to wild-type nibrin. These results indicate that nibrin plays an active role in Atm activation beyond translocating Mre11/Rad50 to the nucleus and that this function requires nibrin-Atm interaction (Cerosaletti, 2006).
DNA double-strand breaks (DSBs) trigger activation of the ATM protein kinase, which coordinates cell-cycle arrest, DNA repair and apoptosis. It is proposed that ATM activation by DSBs occurs in two steps. First, dimeric ATM is recruited to damaged DNA and dissociates into monomers. The Mre11-Rad50-Nbs1 complex (MRN) facilitates this process by tethering DNA, thereby increasing the local concentration of damaged DNA. Notably, increasing the concentration of damaged DNA bypasses the requirement for MRN, and ATM monomers generated in the absence of MRN are not phosphorylated on Ser1981. Second, the ATM-binding domain of Nbs1 is required and sufficient to convert unphosphorylated ATM monomers into enzymatically active monomers in the absence of DNA. This model clarifies the mechanism of ATM activation in normal cells and explains the phenotype of cells from patients with ataxia telangiectasia-like disorder and Nijmegen breakage syndrome (Dupre, 2006).
Nijmegen breakage syndrome (NBS) is characterized by extreme radiation sensitivity, chromosomal instability and cancer. The phenotypes are similar to those of ataxia telangiectasia mutated (ATM) disease, where there is a deficiency in a protein kinase that is activated by DNA damage, indicating that the Nbs and Atm proteins may participate in common pathways. This study reports that Nbs is specifically phosphorylated in response to gamma-radiation, ultraviolet light and exposure to hydroxyurea. Phosphorylation of Nbs mediated by gamma-radiation, but not that induced by hydroxyurea or ultraviolet light, is markedly reduced in ATM cells. In vivo, Nbs is phosphorylated on many serine residues, of which S343, S397 and S615 were phosphorylated by Atm in vitro. At least two of these sites are underphosphorylated in ATM cells. Inactivation of these serines by mutation partially abrogates Atm-dependent phosphorylation. Reconstituting NBS cells with a mutant form of Nbs that cannot be phosphorylated at selected, ATM-dependent serine residues leads to a specific reduction in clonogenic survival after gamma-radiation. Thus, phosphorylation of Nbs by Atm is critical for certain responses of human cells to DNA damage (Wu, 2000).
Mutations in the gene ATM are responsible for the genetic disorder ataxia-telangiectasia (A-T), which is characterized by cerebellar dysfunction, radiosensitivity, chromosomal instability and cancer predisposition. Both the A-T phenotype and the similarity of the ATM protein to other DNA-damage sensors suggests a role for ATM in biochemical pathways involved in the recognition, signalling and repair of DNA double-strand breaks (DSBs). There are strong parallels between the pattern of radiosensitivity, chromosomal instability and cancer predisposition in A-T patients and that in patients with Nijmegen breakage syndrome (NBS). The protein defective in NBS, nibrin (encoded by NBS1), forms a complex with MRE11 and RAD50. This complex localizes to DSBs within 30 minutes after cellular exposure to ionizing radiation (IR) and is observed in brightly staining nuclear foci after a longer period of time. The overlap between clinical and cellular phenotypes in A-T and NBS suggests that ATM and nibrin may function in the same biochemical pathway. This study demonstrates that nibrin is phosphorylated within one hour of treatment of cells with IR. This response is abrogated in A-T cells that either do not express ATM protein or express near full-length mutant protein. ATM physically interacts with and phosphorylates nibrin on serine 343. Phosphorylation of this site appears to be functionally important because mutated nibrin (S343A) does not completely complement radiosensitivity in NBS cells. ATM phosphorylation of nibrin does not affect nibrin-MRE11-RAD50 association as revealed by radiation-induced foci formation. These data provide a biochemical explanation for the similarity in phenotype between A-T and NBS (Gatei, 2000).
The rare diseases ataxia-telangiectasia (AT), caused by mutations in the ATM gene, and Nijmegen breakage syndrome (NBS), with mutations in the p95/nbs1 gene, share a variety of phenotypic abnormalities such as chromosomal instability, radiation sensitivity and defects in cell-cycle checkpoints in response to ionizing radiation. The ATM gene encodes a protein kinase that is activated by ionizing radiation or radiomimetic drugs, whereas p95/nbs1 is part of a protein complex that is involved in responses to DNA double-strand breaks. Because of the similarities between AT and NBS, the functional interactions between ATM and p95/nbs1 were evaluated. Activation of the ATM kinase by ionizing radiation and induction of ATM-dependent responses in NBS cells indicates that p95/nbs1 may not be required for signalling to ATM after ionizing radiation. However, p95/nbs1 is phosphorylated on serine 343 in an ATM-dependent manner in vitro and in vivo after ionizing radiation. A p95/nbs1 construct mutated at the ATM phosphorylation site abrogates an S-phase checkpoint induced by ionizing radiation in normal cells and fails to compensate for this functional deficiency in NBS cells. These observations link ATM and p95/nbs1 in a common signalling pathway and provide an explanation for phenotypic similarities in these two diseases (Lim, 2000).
Nijmegen breakage syndrome (NBS) is characterised by microcephaly, developmental delay, characteristic facial features, immunodeficiency and radiosensitivity. Nbs1, the protein defective in NBS, functions in ataxia telangiectasia mutated protein (ATM)-dependent signalling likely facilitating ATM phosphorylation events. While NBS shares overlapping characteristics with ataxia telangiectasia, it also has features overlapping with ATR-Seckel (ATR: ataxia-telangiectasia and Rad3-related protein) syndrome, a subclass of Seckel syndrome mutated in ATR. Nbs1 also facilitates ATR-dependent phosphorylation. NBS cell lines show a similar defect in ATR phosphorylation of Chk1, c-jun and p-53 in response to UV irradiation- and hydroxyurea (HU)-induced replication stalling. They are also impaired in ubiquitination of FANCD2 after HU treatment, which is ATR dependent. Following HU-induced replication arrest, NBS and ATR-Seckel cells show similarly impaired G2/M checkpoint arrest and an impaired ability to restart DNA synthesis at stalled replication forks. Moreover, NBS cells fail to retain ATR in the nucleus following HU treatment and extraction. These findings suggest that Nbs1 functions in both ATR- and ATM-dependent signalling. It is proposed that the NBS clinical features represent the result of these combined defects (Stiff, 2005).
The Mre11/Rad50/NBS1 (MRN) complex is mutated in inherited genomic instability syndromes featuring cancer predisposition, mental retardation and immunodeficiency. It functions both in DNA double-strand break repair and in controlling the ataxia telangiectasia mutated (ATM) kinase during the response to these lesions. Patients inheriting homozygosity for an NBS1 hypomorphic allele display reduced phosphorylation of signaling factors such as Chk1, but not of chromatin-associated factor H2AX, after stresses that activate the ATM-related kinase, ATR. Therefore, whether MRN has a global controlling role over the ATR kinase was tested through the study of MRN deficiencies generated via RNA interference. MRN is shown to be required for ATR-dependent phosphorylation of structural maintenance of chromosomes 1 (Smc1), which acts within chromatin to ensure sister chromatid cohesion and to effect several DNA damage responses. Novel phenotypes were uncovered, caused by MRN deficiency, that support a functional link between this complex, ATR and Smc1, including hypersensitivity to UV exposure, a defective UV responsive intra-S phase checkpoint and a specific pattern of genomic instability. In addition, certain ATR-dependent responses do not require MRN. These studies demonstrate that there is indeed a controlling role for MRN over the ATR kinase and have established that the downstream events under this control are broad, including both chromatin-associated and diffuse signaling factors, but may not be universal. These studies contribute to an understanding of the central role that MRN plays in damage detection and signaling, which serve to maintain genomic stability and resist neoplastic transformation (Zhong, 2005).
Nijmegen breakage syndrome (NBS) is a rare chromosomal-instability syndrome associated with cancer predisposition, radiosensitivity and radioresistant DNA synthesis-S phase checkpoint deficiency, that results in the failure to suppress DNA replication origins following DNA damage. Approximately 90% of NBS patients are homozygous for the 657del5 allele, a truncating mutation of NBS1 that causes premature termination at codon 219. Because null mutations in MRE11 and RAD50, which encode binding partners of NBS1, are lethal in vertebrates, and mouse Nbs1-null mutants are inviable, the hypothesis was tested that the NBS1 657del5 mutation was a hypomorphic defect. NBS cells were shown to contain the predicted 26-kD amino-terminal protein fragment, NBS1p26, and a 70-kD NBS1 protein (NBS1p70) lacking the native N terminus. The NBSp26 protein is not physically associated with the MRE11 complex, whereas the p70 species is physically associated with it. NBS1p70 is produced by internal translation initiation within the NBS1 mRNA using an open reading frame generated by the 657del5 frameshift. It is proposed that the common NBS1 allele encodes a partially functional protein that diminishes the severity of the NBS phenotype (Maser, 2001).
Nijmegen breakage syndrome (NBS) is a rare autosomal recessive human disease whose clinical features include growth retardation, immunodeficiency, and increased susceptibility to lymphoid malignancies. Cells from NBS patients exhibit gamma-irradiation sensitivity, S-phase checkpoint defects, and genomic instability. Recently, it was demonstrated that this chromosomal breakage syndrome is caused by mutations in the NBS1 gene that result in a total loss of full-length NBS1 expression. This study reports that in contrast to the viability of NBS patients, targeted inactivation of NBS1 in mice leads to early embryonic lethality in utero and is associated with poorly developed embryonic and extraembryonic tissues. Mutant blastocysts showed greatly diminished expansion of the inner cell mass in culture, and this finding suggests that NBS1 mediates essential functions during proliferation in the absence of externally induced damage. Together, these results indicate that the complex phenotypes observed in NBS patients and cell lines may not result from a complete inactivation of NBS1 but may instead result from hypomorphic truncation mutations compatible with cell viability (Zhu, 2001).
Nijmegen breakage syndrome (NBS) is a chromosomal fragility disorder that shares clinical and cellular features with ataxia telangiectasia. Nbs1-null B cells are defective in the activation of ataxia-telangiectasia-mutated (Atm) in response to ionizing radiation, whereas ataxia-telangiectasia- and Rad3-related (Atr)-dependent signalling and Atm activation in response to ultraviolet light, inhibitors of DNA replication, or hypotonic stress are intact. Expression of the main human NBS allele rescues the lethality of Nbs1-/- mice, but leads to immunodeficiency, cancer predisposition, a defect in meiotic progression in females and cell-cycle checkpoint defects that are associated with a partial reduction in Atm activity. The Mre11 interaction domain of Nbs1 is essential for viability, whereas the Forkhead-associated (FHA) domain is required for T-cell and oocyte development and efficient DNA damage signalling. Reconstitution of Nbs1 knockout mice with various mutant isoforms demonstrates the biological impact of impaired Nbs1 function at the cellular and organismal level (Difilippantonio, 2005).
NBS1 forms a complex with MRE11 and RAD50 (MRN) that is proposed to act on the upstream of two repair pathways of DNA double-strand break (DSB), homologous repair (HR) and non-homologous end joining (NHEJ). However, the function of Nbs1 in these processes has not fully been elucidated in mammals due to the lethal phenotype of cells and mice lacking Nbs1. Mouse Nbs1-null embryonic fibroblasts and embryonic stem cells were constructed through the Cre-loxP and sequential gene targeting techniques. Cells lacking Nbs1 display reduced HR of the single DSB in chromosomally integrated substrate, affecting both homology-directed repair (HDR) and single-stranded annealing pathways, and, surprisingly, increased NHEJ-mediated sequence deletion. Moreover, focus formation at DSBs and chromatin recruitment of the Nbs1 partners Rad50 and Mre11 as well as Rad51 and Brca1 are attenuated in these cells, whereas the NHEJ molecule Ku70 binding to chromatin is not affected. These data provide a novel insight into the function of MRN in the branching of DSB repair pathways (Yang, 2006).
Nijmegen breakage syndrome (NBS) is a chromosomal-instability syndrome associated with cancer predisposition, radiosensitivity, microcephaly, and growth retardation. The NBS gene product, NBS1, is a component of the MRE11-RAD50-NBS1 (MRN) complex, a central player associated with double strand break (DSB) repair. In response to radiation, NBS1 is phosphorylated by ATM, and the MRN complex relocalizes to form punctate nuclear foci for DNA repair. NBS1 controls both the nuclear localization of the MRN complexes and radiation-induced focus formation. The KPNA2 (importin alpha1) is important for the normal nuclear localization of the MRN complex and its proper formation of the nuclear foci. KPNA2 is the only member of the importin alpha family that physically interacts with NBS1, and the KPNA2-mediated nucleus localization sequence (NLS) is mapped to amino acid residues 461-467 of NBS1 that is sufficient for both the interaction with KPNA2 and the proper nuclear localization. Inhibition of KPNA2 or blockage of the KPNA2 interaction with NBS1 results in a reduction of radiation-induced nuclear focus accumulation, DSB repair, and cell cycle checkpoint signaling of NBS1. Collectively, these results strongly suggest that an interaction with KPNA2 contributes to nuclear localization and multiple tumor suppression functions of the NBS1 complex (Tseng, 2005).
DNA palindromes are rare in humans but are associated with meiosis-specific translocations. The conserved Mre11/Rad50/Nbs1 (MRN) complex is likely directly involved in processing palindromes through the homologous recombination pathway of DNA repair. Using the fission yeast Schizosaccharomyces pombe as a model system, it is shown that a 160-bp palindrome (M-pal) is a meiotic recombination hotspot and is preferentially eliminated by gene conversion. Importantly, this hotspot depends on the MRN complex for full activity and reveals a new pathway for generating meiotic DNA double-strand breaks (DSBs), separately from the Rec12 (ortholog of Spo11) pathway. MRN-dependent DSBs are formed at or near the M-pal in vivo, and in contrast to the Rec12-dependent breaks, they appear early, during premeiotic replication. Analysis of mrn mutants indicates that the early DSBs are generated by the MRN nuclease activity, demonstrating the previously hypothesized MRN-dependent breakage of hairpins during replication. These studies provide a genetic and physical basis for frequent translocations between palindromes in human meiosis and identify a conserved meiotic process that constantly selects against palindromes in eukaryotic genomes (Farah, 2005).
In mammals, the ATM and ATR protein kinases function as critical regulators of the cellular DNA damage response. The checkpoint functions of ATR and ATM are mediated, in part, by a pair of checkpoint effector kinases termed Chk1 and Chk2. In mammalian cells, evidence has been presented that Chk1 is devoted to the ATR signaling pathway and is modified by ATR in response to replication inhibition and UV-induced damage, whereas Chk2 functions primarily through ATM in response to ionizing radiation (IR), suggesting that Chk2 and Chk1 might have evolved to channel the DNA damage signal from ATM and ATR, respectively. The ATR-Chk1 and ATM-Chk2 pathways are not parallel branches of the DNA damage response pathway but instead show a high degree of cross-talk and connectivity. ATM does in fact signal to Chk1 in response to IR. Phosphorylation of Chk1 on Ser-317 in response to IR is ATM-dependent. Functional NBS1 is required for phosphorylation of Chk1, indicating that NBS1 might facilitate the access of Chk1 to ATM at the sites of DNA damage. Abrogation of Chk1 expression by RNA interference results in defects in IR-induced S and G(2)/M phase checkpoints; however, the overexpression of phosphorylation site mutant (S317A, S345A or S317A/S345A double mutant) Chk1 fails to interfere with these checkpoints. Surprisingly, the kinase-dead Chk1 (D130A) also fails to abrogate the S and G(2) checkpoint through any obvious dominant negative effect toward endogenous Chk1. Therefore, further studies will be required to assess the contribution made by phosphorylation events to Chk1 regulation. Overall, the data presented in the study challenge the model in which Chk1 functions downstream from ATR only and does indicate that ATM signals to Chk1. In addition, this study also demonstrates that Chk1 is essential for IR-induced inhibition of DNA synthesis and the G(2)/M checkpoint (Gatei, 2003).
The checkpoint kinase Chk2 has a key role in delaying cell cycle progression in response to DNA damage. Upon activation by low-dose ionizing radiation (IR), which occurs in an ataxia telangiectasia mutated (ATM)-dependent manner, Chk2 can phosphorylate the mitosis-inducing phosphatase Cdc25C on an inhibitory site, blocking entry into mitosis, and p53 on a regulatory site, causing G(1) arrest. This study shows that the ATM-dependent activation of Chk2 by gamma-radiation requires Nbs1, the gene product involved in the Nijmegen breakage syndrome (NBS), a disorder that shares with AT a variety of phenotypic defects including chromosome fragility, radiosensitivity, and radioresistant DNA synthesis. Thus, whereas in normal cells Chk2 undergoes a time-dependent increased phosphorylation and induction of catalytic activity against Cdc25C, in NBS cells null for Nbs1 protein, Chk2 phosphorylation and activation are both defective. Importantly, these defects in NBS cells can be complemented by reintroduction of wild-type Nbs1, but neither by a carboxy-terminal deletion mutant of Nbs1 at amino acid 590, unable to form a complex with and to transport Mre11 and Rad50 in the nucleus, nor by an Nbs1 mutated at Ser343 (S343A), the ATM phosphorylation site. Chk2 nuclear expression is unaffected in NBS cells, hence excluding a mislocalization as the cause of failed Chk2 activation in Nbs1-null cells. Interestingly, the impaired Chk2 function in NBS cells correlates with the inability, unlike normal cells, to stop entry into mitosis immediately after irradiation, a checkpoint abnormality that can be corrected by introduction of the wild-type but not the S343A mutant form of Nbs1. Altogether, these findings underscore the crucial role of a functional Nbs1 complex in Chk2 activation and suggest that checkpoint defects in NBS cells may result from the inability to activate Chk2 (Buscemi, 2001).
The Mre11.Rad50.Nbs1 (MRN) complex binds DNA double strand breaks to repair DNA and activate checkpoints. MRN deficiency occurs in three of seven colon carcinoma cell lines of the NCI Anticancer Drug Screen. To study the involvement of MRN in replication-mediated DNA double strand breaks, checkpoint responses were examined to camptothecin, which induces replication-mediated DNA double strand breaks after replication forks collide with topoisomerase I cleavage complexes. MRN-deficient cells were deficient for Chk2 activation, whereas Chk1 activation is independent of MRN. Chk2 activation is ataxia telangiectasia mutated (ATM)-dependent and associated with phosphorylation of Mre11 and Nbs1. Mre11 complementation in MRN-deficient HCT116 cells restores Chk2 activation as well as Rad50 and Nbs1 levels. Conversely, Mre11 down-regulation by small interference RNA (siRNA) in HT29 cells inhibits Chk2 activation and down-regulated Nbs1 and Rad50. Proteasome inhibition also restores Rad50 and Nbs1 levels in HCT116 cells suggesting that Mre11 stabilizes Rad50 and Nbs1. Chk2 activation was also defective in three of four MRN-proficient colorectal cell lines because of low Chk2 levels. Thus, six of seven colon carcinoma cell lines from the NCI Anticancer Drug Screen are functionally Chk2-deficient in response to replication-mediated DNA double strand breaks. It is proposed that Mre11 stabilizes Nbs1 and Rad50 and that MRN activates Chk2 downstream from ATM in response to replication-mediated DNA double strand breaks. Chk2 deficiency in HCT116 is associated with defective S-phase checkpoint, prolonged G2 arrest, and hypersensitivity to camptothecin. The high frequency of MRN and Chk2 deficiencies may contribute to genomic instability and therapeutic response to camptothecins in colorectal cancers (Takemura, 2006).
Structural maintenance of chromosomes (SMC) proteins play important roles in sister chromatid cohesion, chromosome condensation, sex-chromosome dosage compensation, and DNA recombination and repair. Protein complexes containing heterodimers of the Smc1 and Smc3 proteins have been implicated specifically in both sister chromatid cohesion and DNA recombination. The protein kinase Atm phosphorylates Smc1 protein after ionizing irradiation. Atm phosphorylates Smc1 on serines 957 and 966 in vitro and in vivo, and expression of an Smc1 protein mutated at these phosphorylation sites abrogates the ionizing irradiation-induced S phase cell cycle checkpoint. Optimal phosphorylation of these sites in Smc1 after ionizing irradiation also requires the presence of the Atm substrates Nbs1 and Brca1. These same sites in Smc1 are phosphorylated after treatment with UV irradiation or hydroxyurea in an Atm-independent manner, thus demonstrating that another kinase must be involved in responses to these cellular stresses. Yeast containing hypomorphic mutations in SMC1 and human cells overexpressing Smc1 mutated at both of these phosphorylation sites exhibit decreased survival following ionizing irradiation. These results demonstrate that Smc1 participates in cellular responses to DNA damage and link Smc1 to the Atm signal transduction pathway (Kim, 2002).
Structural maintenance of chromosomes (SMC) proteins (SMC1, SMC3) are evolutionarily conserved chromosomal proteins that are components of the cohesin complex, necessary for sister chromatid cohesion. These proteins may also function in DNA repair. SMC1 is a component of the DNA damage response network that functions as an effector in the ATM/NBS1-dependent S-phase checkpoint pathway. SMC1 associates with BRCA1 and is phosphorylated in response to IR in an ATM- and NBS1-dependent manner. Using mass spectrometry, it has been established that ATM phosphorylates S957 and S966 of SMC1 in vivo. Phosphorylation of S957 and/or S966 of SMC1 is required for activation of the S-phase checkpoint in response to IR. The phosphorylation of NBS1 (Nijmegen breakage syndrome gene product is a part of the hMre11 complex, a central player associated with double-strand break repair) by ATM is required for the phosphorylation of SMC1, establishing the role of NBS1 as an adaptor in the ATM/NBS1/SMC1 pathway. The ATM/CHK2/CDC25A pathway is also involved in the S-phase checkpoint activation, but this pathway is intact in NBS cells. These results indicate that the ATM/NBS1/SMC1 pathway is a separate branch of the S-phase checkpoint pathway, distinct from the ATM/CHK2/CDC25A branch. Therefore, this work establishes the ATM/NBS1/SMC1 branch, and provides a molecular basis for the S-phase checkpoint defect in NBS cells (Yazdi, 2002).
The ATM protein kinase is activated by intermolecular autophosphorylation in response to DNA damage and initiates cellular signaling pathways that facilitate cell survival and reduce chromosomal breakage. NBS1 and BRCA1 are required for the recruitment of previously activated ATM to the sites of DNA breaks after ionizing irradiation, and this recruitment is required for the phosphorylation of structural maintenance of chromosome protein 1 (SMC1) by ATM. To explore the functional importance of SMC1 phosphorylation, murine cells were generated, in which the two damage-induced phosphorylation sites in SMC1 are mutated. Although these cells demonstrate normal phosphorylation and focus formation of ATM, NBS1, and BRCA1 proteins after IR, they exhibit a defective S-phase checkpoint, decreased survival, and increased chromosomal aberrations after DNA damage. These observations suggest that many of the abnormal stress responses seen in cells lacking ATM, NBS1, or BRCA1 result from a failure of ATM migration to sites of DNA breaks and a resultant lack of SMC1 phosphorylation (Kitagawa, 2004).
Transactivation-transformation domain-associated protein (TRRAP) is a component of several multiprotein histone acetyltransferase (HAT) complexes implicated in transcriptional regulation. TRRAP was shown to be required for the mitotic checkpoint and normal cell cycle progression. MRE11, RAD50, and NBS1 (product of the Nijmegan breakage syndrome gene) form the MRN complex that is involved in the detection, signaling, and repair of DNA double-strand breaks (DSBs). By using double immunopurification, mass spectrometry, and gel filtration, the stable association of TRRAP with the MRN complex is described. The TRRAP-MRN complex is not associated with any detectable HAT activity, while the isolated other TRRAP complexes, containing either GCN5 or TIP60, are. TRRAP-depleted extracts show a reduced nonhomologous DNA end-joining activity in vitro. Importantly, small interfering RNA knockdown of TRRAP in HeLa cells or TRRAP knockout in mouse embryonic stem cells inhibit the DSB end-joining efficiency and the precise nonhomologous end-joining process, further suggesting a functional involvement of TRRAP in the DSB repair processes. Thus, TRRAP may function as a molecular link between DSB signaling, repair, and chromatin remodeling (Robert, 2006).
Human MDC1/NFBD1 has been found to interact with key players of the DNA-damage response machinery. This study identifies and describes a functional homologue of MDC1/ NFBD1 in Mus musculus. The mouse homologue, mMDC1, retains the key motifs identified in the human protein and in response to ionizing radiation forms foci that co-localize with the MRE11-RAD50-NBS1 (MRN) complex and factors such as gammaH2AX and 53BP1. In addition, mMDC1 is associated with DNA damage sites generated during meiotic recombination as well as the X and Y chromosomes during the late stages of meiotic prophase I. Finally, whereas MDC1 shows strong colocalization with the MRN complex in response to DNA damage it does not co-localize with the MRN complex on replicating chromatin. These data suggest that mMDC1 is a marker for both exogenously and endogenously generated DNA double-stranded breaks and that its interaction with the MRN complex is initiated exclusively by DNA damage (A. C. Lee, 2005).
Nijmegen breakage syndrome (NBS) is a chromosomal instability syndrome associated with cancer predisposition, radiosensitivity, microcephaly, and growth retardation. The NBS gene product, NBS1 (p95) or nibrin, is a part of the hMre11 complex, a central player associated with double strand break repair. c-Myc directly activates NBS1 expression. This study shows that constitutive expression of NBS1 in Rat1a and HeLa cells induces/enhances their transformation. Repression of endogenous NBS1 levels using short interference RNA reduces the transformation activity of two tumor cell lines. Increased NBS1 expression is observed in 40-52% of non-small cell lung carcinoma, hepatoma, and esophageal cancer samples. NBS1 overexpression stimulates phosphatidylinositol (PI) 3-kinase activity, leading to increased phosphorylation levels of Akt and its downstream targets such as glycogen synthase kinase 3beta and mammalian target of rapamycin in different cell lines and tumor samples. Transformation induced by NBS1 overexpression can be inhibited by a PI3-kinase inhibitor (LY294002). Repression of endogenous Akt expression by short interference RNA decreases the transformation activity of Rat1a cells overexpressing NBS1. These results indicate that overexpression of NBS1 is an oncogenic event that contributes to transformation through the activation of PI3-kinase/Akt (Chen, 2005).
Nijmegen breakage syndrome (NBS) is a rare autosomal recessive disorder characterized by predisposition to hematopoietic malignancy, cell-cycle checkpoint defects, and ionizing radiation sensitivity. NBS is caused by a hypomorphic mutation of the NBS1 gene, encoding nibrin, which forms a protein complex with Mre11 and Rad50, both involved in DNA repair. Nibrin localizes to chromosomal sites of class switching, and B cells from NBS patients show an enhanced presence of microhomologies at the sites of switch recombination. Because nibrin is crucial for embryonic survival, direct demonstration by targeted deletion that nibrin functions in class switch recombination has been lacking. This study shows by cell-type-specific conditional inactivation of Nbn, the murine homologue of NBS1, that nibrin plays a role in the repair of gamma-irradiation damage, maintenance of chromosomal stability, and the recombination of Ig constant region genes in B lymphocytes (Kracker, 2005).
Mre11, Rad50, and Nbs1 form an evolutionarily conserved protein complex (Mre11-Rad50-Nbs1, MRN) that has been proposed to function as a DNA damage sensor. Hypomorphic mutations in Mre11 and Nbs1 result in the human ataxia-telangiectasia-like disorder and Nijmegen breakage syndrome (NBS), respectively. In contrast, complete inactivation of Mre11, Rad50, or Nbs1 leads to early embryonic lethality, suggesting that the hypomorphic mutations may fail to reveal some of the essential functions of MRN. This study uses Cre-loxP-mediated recombination to restrict Nbs1 deletion to B lymphocytes. Disruption of Nbs1 results in the accumulation of high levels of spontaneous DNA damage, impaired proliferation, and chromosomal endoreduplication. Moreover, Ig class-switch recombination (CSR) is diminished in Nbs1-deficient B cells. The CSR defect is B cell-intrinsic, independent of switch-region transcription, and a consequence of inefficient recombination at the DNA level. These findings reveal that Nbs1 is critical for efficient Ig CSR and maintenance of the integrity of chromosomal structure and number (Reina-San-Martin, 2005).
The promyelocytic leukemia (PML) nuclear body (NB) is a dynamic subnuclear compartment that is implicated in tumor suppression, as well as in the transcription, replication, and repair of DNA. PML NB number can change during the cell cycle, increasing in S phase and in response to cellular stress, including DNA damage. Although topological changes in chromatin after DNA damage may affect the integrity of PML NBs, the molecular or structural basis for an increase in PML NB number has not been elucidated. This study demonstrates that after DNA double-strand break induction, the increase in PML NB number is based on a biophysical process, as well as ongoing cell cycle progression and DNA repair. PML NBs increase in number by a supramolecular fission mechanism similar to that observed in S-phase cells, and which is delayed or inhibited by the loss of function of NBS1, ATM, Chk2, and ATR kinase. Therefore, an increase in PML NB number is an intrinsic element of the cellular response to DNA damage (Dellaire, 2006).
Search PubMed for articles about Drosophila Nijmegen breakage syndrome
Adams, K. E., Medhurst, A. L., Dart, D. A. and Lakin, N. D. (2006). Recruitment of ATR to sites of ionising radiation-induced DNA damage requires ATM and components of the MRN protein complex. Oncogene 25(28): 3894-904. 16474843
Becker, E., Meyer, V., Madaoui, H. and Guerois, R. (2006). Detection of a tandem BRCT in Nbs1 and Xrs2 with functional implications in the DNA damage response. Bioinformatics 22(11): 1289-92. 16522671
Bi, X., Wei, S. C. and Rong, Y. (2004). Telomere protection without a telomerase: the role of ATM and Mre11 in Drosophila telomere maintenance. Curr. Biol. 14: 1348-53. 15296751
Bi, X., Srikanta, D., Fanti, L., Pimpinelli, S., Badugu, R., Kellum, R. and Rong, Y. S. (2005). Drosophila ATM and ATR checkpoint kinases control partially redundant pathways for telomere maintenance. Proc. Natl. Acad. Sci. 102(42): 15167-72. 16203987
Brodsky, M. H., Sekelsky, J. J., Tsang, G., Hawley, R. S. and Rubin, G. M. (2002). mus304 encodes a novel DNA damage checkpoint protein required during Drosophila development. Genes Dev. 14(6): 666-78. 10733527
Buscemi, G., et al. (2001). Chk2 activation dependence on Nbs1 after DNA damage. Mol. Cell. Biol. 21: 5214-5222. 11438675
Carney, J. P., et al. (1998) The hMre11/hRad50 protein complex and Nijmegen breakage syndrome: linkage of double-strand break repair to the cellular DNA damage response. Cell 93: 477-486. 9590181
Cenci, G., Ciapponi, L. and GATTI, M. (2005). The mechanism of telomere protection: a comparison between Drosophila and humans. Chromosoma 114: 135-145. 16012858
Cerosaletti, K. M. and Concannon, P. (2003). Nibrin forkhead-associated domain and breast cancer C-terminal domain are both required for nuclear focus formation and phosphorylation. J. Biol. Chem. 278(24): 21944-51. 12679336
Cerosaletti, K., and Concannon, P. (2004). Independent roles for nibrin and Mre11-Rad50 in the activation and function of Atm. J. Biol. Chem. 279: 38813-38819. 15234984
Cerosaletti, K., Wright, J. and Concannon, P. (2006). Active role for nibrin in the kinetics of atm activation. Mol. Cell. Biol. 26: 1691-1699. 16478990
Chai, W., Sfeir, A. J., Hoshiyama, H., Shay, J. W. and Wright, W. E. (2006). The involvement of the Mre11/Rad50/Nbs1 complex in the generation of G-overhangs at human telomeres. EMBO Rep. 7(2): 225-30. 16374507
Chen, Y. C., et al. (2005). Overexpression of NBS1 contributes to transformation through the activation of phosphatidylinositol 3-kinase/Akt. J. Biol. Chem. 280(37): 32505-11. 16036916
Chiang, Y. C., et al. (2003). c-Myc directly regulates the transcription of the NBS1 gene involved in DNA double-strand break repair. J. Biol. Chem. 278(21): 19286-91. 12637527
Ciapponi, L. X., Cenci, G. X., Ducau, J. X., Flores, C. X., Johnson-Schlitz, D. X., Gorski, M. M. X., Engels, W. and Gatti, M. (2004). The Drosophila Mre11/Rad50 complex is required to prevent both telomeric fusion and chromosome breakage. Curr. Biol. 14: 1360-6. 15296753
Ciapponi, L., Cenci, G. and Gatti M. (2006). The Drosophila Nbs protein functions in multiple pathways for the maintenance of genome stability. Genetics 173(3): 1447-54. 16648644
d'Adda di Fagagna, F., Teo, S. H. and Jackson, S. P. (2004). Functional links between telomeres and proteins of the DNA-damage response. Genes Dev. 18: 1781-1799. 15289453
Dellaire, G., Ching, R. W., Ahmed, K., Jalali, F., Tse, K. C.K., Bristow, R. G., Bazett-Jones, D. P. (2006). Promyelocytic leukemia nuclear bodies behave as DNA damage sensors whose response to DNA double-strand breaks is regulated by NBS1 and the kinases ATM, Chk2, and ATR. J. Cell Biol. 175: 55-66. 17030982
Difilippantonio, S., et al. (2005). Role of Nbs1 in the activation of the Atm kinase revealed in humanized mouse models. Nat. Cell Biol. 7: 675-685. 15965469
Digweed, M., and Sperling, K. (2004). Nijmegen breakage syndrome: clinical manifestation of defective response to DNA double-strand breaks. DNA Rep. 3: 1207-1217. 15279809
Dong, Z., Zhong, Q. and Chen, P. L. (1999). The Nijmegen breakage syndrome protein is essential for Mre11 phosphorylation upon DNA damage. J. Biol. Chem. 274(28): 19513-6. 10391882
Dupre, A., Boyer-Chatenet, L. and Gautier J. (2006). Two-step activation of ATM by DNA and the Mre11-Rad50-Nbs1 complex. Nat. Struct. Mol. Biol. 13(5): 451-7. 16622404
Farah, J. A., Cromie, G., Steiner, W. W. and Smith, G. R. (2005). A novel recombination pathway initiated by the Mre11/Rad50/Nbs1 complex eliminates palindromes during meiosis in Schizosaccharomyces pombe. Genetics 169(3): 1261-74. 15654094
Gatei, M., et al. (2000). ATM-dependent phosphorylation of nibrin in response to radiation exposure. Nat. Genet. 25(1): 115-9. 10802669
Gatei, M., et al. (2003). Ataxia-telangiectasia-mutated (ATM) and NBS1-dependent phosphorylation of Chk1 on Ser-317 in response to ionizing radiation. J. Biol. Chem. 278(17): 14806-11. 12588868
Iijima, K., Komatsu, K., Matsuura, S. and Tauchi, H. (2004). The Nijmegen breakage syndrome gene and its role in genome stability. Chromosoma 113: 53-61. 15258809
Kim, S. T., Xu, B. and Kastan, M. B. (2002). Involvement of the cohesin protein, Smc1, in Atm-dependent and independent responses to DNA damage. Genes Dev. 16(5): 560-70. 11877376
Kitagawa, R., Bakkenist, C. J., McKinnon, P. J. and Kastan, M. B. (2004). Phosphorylation of SMC1 is a critical downstream event in the ATM-NBS1-BRCA1 pathway. Genes Dev. 18(12): 1423-38. 15175241
Kobayashi, J., et al. (2002). NBS1 localizes to gamma-H2AX foci through interaction with the FHA/BRCT domain. Curr Biol 12: 1846-1851. 12419185
Kracker, S., et al. (2005). Nibrin functions in Ig class-switch recombination. Proc. Natl. Acad. Sci. 102(5): 1584-9. 15668383
Lee, A. C., Fernandez-Capetillo, O., Pisupati, V., Jackson, S. P. and Nussenzweig, A. (2005). Specific association of mouse MDC1/NFBD1 with NBS1 at sites of DNA-damage. Cell Cycle 4(1):177-82. 15611643
Lee, J. H. and Paull, T. T. (2004). Direct activation of the ATM protein kinase by the Mre11/Rad50/Nbs1 complex. Science 304(5667): 93-6. 15064416
Lee, J.-H. and Paull, T. T. (2005). ATM activation by DNA double-strand breaks through the Mre11-Rad50-Nbs1 complex. Science 308: 551-554. 15790808
Lee, J.-H., Lim, D.-S. (2006). Dual role of Nbs1 in the ataxia telangiectasia mutated-dependent DNA damage response. FEBS J 273: 1630-1636. 16623700
Lim, D. S., et al. (2000). ATM phosphorylates p95/nbs1 in an S-phase checkpoint pathway. Nature 404(6778): 613-7. 10766245
Maser, R. S., Zinkel, R. and Petrini, J. H. (2001). An alternative mode of translation permits production of a variant NBS1 protein from the common Nijmegen breakage syndrome allele. Nat. Genet. 27: 417-421. 11279524
Moreno-Herrero, F., et al. (2005). Mesoscale conformational changes in the DNA-repair complex Rad50/Mre11/Nbs1 upon binding DNA. Nature 437(7057): 440-3. 16163361
Oikemus, S. R., Queiroz-Machado, J., Lai, K., McGinnis, N., Sunkel, C. and Brodsky, M. H. (2006). Epigenetic telomere protection by Drosophila DNA damage response pathways. PLoS Genet. 2(5): e71. 16710445
Reina-San-Martin, B., Nussenzweig, M. C., Nussenzweig, A. and Difilippantonio, S. (2005). Genomic instability, endoreduplication, and diminished Ig class-switch recombination in B cells lacking Nbs1. Proc. Natl. Acad. Sci. 102(5): 1590-5. 15668392
Ritchie, K. B. and Petes, T. D. (2000). The Mre11p/Rad50p/Xrs2p complex and the Tel1p function in a single pathway for telomere maintenance in yeast. Genetics 155: 475-479. 10790418
Robert, F., et al. (2006). The transcriptional histone acetyltransferase cofactor TRRAP associates with the MRN repair complex and plays a role in DNA double-strand break repair. Mol. Cell. Biol. 26(2): 402-12. 16382133
Robison, J. G., Lu, L., Dixon, K. and Bissler, J. J. (2005). DNA lesion-specific co-localization of the Mre11/Rad50/Nbs1 (MRN) complex and replication protein A (RPA) to repair foci. J. Biol. Chem. 280(13): 12927-34. 15653682
Shiloh, Y. (2003). ATM and related protein kinases: Safeguarding genome integrity. Nat. Rev. Cancer 3: 155-168. 12612651
Silverman, J., Takai, H., Buonomo, S. B., Eisenhaber, F. and de Lange T. (2004). Human Rif1, ortholog of a yeast telomeric protein, is regulated by ATM and 53BP1 and functions in the S-phase checkpoint. Genes Dev. 18(17): 2108-19. 15342490
Stewart, G. S., et al. (1999). The DNA double-strand break repair gene hMRE11 is mutated in individuals with an ataxia-telangiectasia-like disorder. Cell 99: 577-587. 10612394
Stiff, T., et al. (2005). Nbs1 is required for ATR-dependent phosphorylation events. EMBO J. 24: 199-208. 15616588
Stracker, T. H., et al. (2004). The Mre11 complex and the metabolism of chromosome breaks: the importance of communicating and holding things together. DNA Rep. 3: 845-854. 15279769
Takemura, H., et al. (2006). Defective Mre11-dependent activation of Chk2 by ataxia telangiectasia mutated in colorectal carcinoma cells in response to replication-dependent DNA double strand breaks. J. Biol. Chem. 281(41): 30814-23. 16905549
Tseng, S. F., Chang, C. Y., Wu, K. J. and Teng, S. C. (2005). Importin KPNA2 is required for proper nuclear localization and multiple functions of NBS1. J. Biol. Chem. 280(47): 39594-600. 16188882
Uziel, T., et al. (2003). Requirement of the MRN complex for ATM activation by DNA damage. EMBO J. 22: 5612-5621. 14532133
Varon, R., et al. (1998). Nibrin, a novel DNA double-strand break repair protein, is mutated in Nijmegen breakage syndrome. Cell 93(3): 467-76. 9590180
Wu, X., et al. (2000). ATM phosphorylation of Nijmegen breakage syndrome protein is required in a DNA damage response. Nature 405: 477-482. 10839545
Yang, Y. G., et al. (2006). Conditional deletion of Nbs1 in murine cells reveals its role in branching repair pathways of DNA double-strand breaks. EMBO J. 25(23): 5527-38. 17082765
Yazdi, P. T., Wang, Y., Zhao, S., Patel, N., Lee, E. Y. and Qin, J. (2002). SMC1 is a downstream effector in the ATM/NBS1 branch of the human S-phase checkpoint. Genes Dev. 16(5): 571-82. 11877377
You, Z., Chahwan, C., Bailis, J., Hunter, T. and Russell, P. (2005). ATM activation and its recruitment to damaged DNA require binding to the C terminus of Nbs1. Mol. Cell. Biol. 25(13): 5363-79. 15964794
Zhang, Y., et al. (2005). NBS1 knockdown by small interfering RNA increases ionizing radiation mutagenesis and telomere association in human cells. Cancer Res. 65: 5544-5553. 15994926
Zhao, S., et al. (2000). Functional link between ataxia-telangiectasia and Nijmegen breakage syndrome gene products. Nature 405: 473-477. 10839544
Zhong, H., et al. (2005). Rad50 depletion impacts upon ATR-dependent DNA damage responses. Hum. Mol. Genet. 14: 2685-2693. 16087684
Zhu, J., Petersen, S., Tessarollo, L. and Nussenzweig, A. (2001). Targeted disruption of the Nijmegen breakage syndrome gene NBS1 leads to early embryonic lethality in mice. Curr. Biol. 11: 105-109. 11231126
Zhu, X. D., et al. (2000). Cell-cycle-regulated association of RAD50/MRE11/NBS1 with TRF2 and human telomeres. Nat. Genet. 25: 347-352. 10888888
date revised: 30 December 2006
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